Compositions and methods for TCR reprogramming using fusion proteins

ABSTRACT

Provided herein are T-cell receptor (TCR) fusion proteins (TFPs), T-cells engineered to express one or more TFPs, and methods of use thereof for the treatment of diseases, including cancer.

CROSS REFERENCE

This application is a continuation of U.S. application Ser. No.16/222,846, filed Dec. 17, 2018, which is a divisional of U.S.application Ser. No. 15/888,897, filed on Feb. 5, 2018, now issued asU.S. Pat. No. 10,208,285 on Feb. 19, 2019, which is a continuation ofInternational Application No. PCT/US2017/055628, filed Oct. 6, 2017,which claims the benefit of U.S. Provisional Application No. 62/405,551,filed Oct. 7, 2016, and U.S. Provisional Application No. 62/510,108,filed May 23, 2017, each of which is incorporated herein by reference inits entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 10, 2020, isnamed “48538702303_SL.txt” and is 113,197 bytes in size.

BACKGROUND OF THE INVENTION

Most patients with late-stage solid tumors are incurable with standardtherapy. In addition, traditional treatment options often have seriousside effects. Numerous attempts have been made to engage a patient'simmune system for rejecting cancerous cells, an approach collectivelyreferred to as cancer immunotherapy. However, several obstacles make itrather difficult to achieve clinical effectiveness. Although hundreds ofso-called tumor antigens have been identified, these are often derivedfrom self and thus can direct the cancer immunotherapy against healthytissue, or are poorly immunogenic. Furthermore, cancer cells usemultiple mechanisms to render themselves invisible or hostile to theinitiation and propagation of an immune attack by cancerimmunotherapies.

Recent developments using chimeric antigen receptor (CAR) modifiedautologous T-cell therapy, which relies on redirecting geneticallyengineered T-cells to a suitable cell-surface molecule on cancer cells,show promising results in harnessing the power of the immune system totreat B cell malignancies (see, e.g., Sadelain et al., Cancer Discovery3:388-398 (2013)). The clinical results with CD-19-specific CAR T-cells(called CTL019) have shown complete remissions in patients sufferingfrom chronic lymphocytic leukemia (CLL) as well as in childhood acutelymphoblastic leukemia (ALL) (see, e.g., Kalos et al., Sci Transl Med3:95ra73 (2011), Porter et al., NEJM 365:725-733 (2011), Grupp et al.,NEJM 368:1509-1518 (2013)). An alternative approach is the use of T-cellreceptor (TCR) alpha and beta chains selected for a tumor-associatedpeptide antigen for genetically engineering autologous T-cells. TheseTCR chains will form complete TCR complexes and provide the T-cells witha TCR for a second defined specificity. Encouraging results wereobtained with engineered autologous T-cells expressing NY-ESO-1-specificTCR alpha and beta chains in patients with synovial carcinoma.

Besides the ability of genetically modified T-cells expressing a CAR ora second TCR to recognize and destroy respective target cells invitro/ex vivo, successful patient therapy with engineered T-cellsrequires the T-cells to be capable of strong activation, expansion,persistence overtime, and, in case of relapsing disease, to enable a‘memory’ response. High and manageable clinical efficacy of CAR T-cellsis currently limited to BCMA- and CD-19-positive B cell malignancies andto NY-ESO-1-peptide expressing synovial sarcoma patients expressingHLA-A2. There is a clear need to improve genetically engineered T-cellsto more broadly act against various human malignancies. Described hereinare novel fusion proteins of TCR subunits, including CD3 epsilon,CD3gamma and CD3 delta, and of TCR alpha and TCR beta chains withbinding domains specific for cell surface antigens that have thepotential to overcome limitations of existing approaches. Describedherein are novel fusion proteins that more efficiently kill target cellsthan CARs, but release comparable or lower levels of pro-inflammatorycytokines. These fusion proteins and methods of their use represent anadvantage for T-cell receptor (TCR) fusion proteins (TFPs) relative toCARs because elevated levels of these cytokines have been associatedwith dose-limiting toxicities for adoptive CAR-T therapies.

SUMMARY OF THE INVENTION

Provided herein are T-cell receptor (TCR) fusion proteins (TFPs),T-cells engineered to express one or more TFPs, and methods of usethereof for the treatment of diseases.

In one aspect, provided herein is an isolated recombinant nucleic acidmolecule encoding a T-cell receptor (TCR) fusion protein (TFP)comprising a TCR subunit and a human or humanized antibody domaincomprising an anti-mesothelin binding domain.

In one aspect, provided herein is an isolated recombinant nucleic acidmolecule encoding a T-cell receptor (TCR) fusion protein (TFP)comprising a TCR subunit comprising at least a portion of a TCRextracellular domain, and a TCR intracellular domain comprising astimulatory domain from an intracellular signaling domain of CD3epsilon; and a human or humanized antibody domain comprising an antigenbinding domain wherein the TCR subunit and the antibody domain areoperatively linked, and wherein the TFP incorporates into a TCR whenexpressed in a T-cell.

In one aspect, provided herein is an isolated recombinant nucleic acidmolecule encoding a T-cell receptor (TCR) fusion protein (TFP)comprising a TCR subunit comprising at least a portion of a TCRextracellular domain, and a TCR intracellular domain comprising astimulatory domain from an intracellular signaling domain of CD3 gamma;and a human or humanized antibody domain comprising an antigen bindingdomain wherein the TCR subunit and the antibody domain are operativelylinked, and wherein the TFP incorporates into a TCR when expressed in aT-cell.

In one aspect, provided herein is an isolated recombinant nucleic acidmolecule encoding a T-cell receptor (TCR) fusion protein (TFP)comprising a TCR subunit comprising at least a portion of a TCRextracellular domain, and a TCR intracellular domain comprising astimulatory domain from an intracellular signaling domain of CD3 delta;and a human or humanized antibody domain comprising an antigen bindingdomainwherein the TCR subunit and the antibody domain are operativelylinked, and wherein the TFP incorporates into a TCR when expressed in aT-cell.

In one aspect, provided herein is an isolated recombinant nucleic acidmolecule encoding a T-cell receptor (TCR) fusion protein (TFP)comprising a TCR subunit comprising at least a portion of a TCRextracellular domain, and a TCR intracellular domain comprising astimulatory domain from an intracellular signaling domain of TCR alpha;and a human or humanized antibody domain comprising an antigen bindingdomain wherein the TCR subunit and the antibody domain are operativelylinked, and wherein the TFP incorporates into a TCR when expressed in aT-cell.

In one aspect, provided herein is an isolated recombinant nucleic acidmolecule encoding a T-cell receptor (TCR) fusion protein (TFP)comprising a TCR subunit comprising at least a portion of a TCRextracellular domain, and a TCR intracellular domain comprising astimulatory domain from an intracellular signaling domain of TCR beta;and a human or humanized antibody domain comprising an antigen bindingdomain wherein the TCR subunit and the antibody domain are operativelylinked, and wherein the TFP incorporates into a TCR when expressed in aT-cell.

In one aspect, provided herein is an isolated recombinant nucleic acidmolecule encoding a T-cell receptor (TCR) fusion protein (TFP)comprising a TCR subunit and a human or humanized antibody domaincomprising an antigen binding domain that is an anti-mesothelin bindingdomain.

In some instances, the TCR subunit and the antibody domain areoperatively linked. In some instances, the TFP incorporates into a TCRwhen expressed in a T-cell. In some instances, the encoded antigenbinding domain is connected to the TCR extracellular domain by a linkersequence. In some instances, the encoded linker sequence comprises(G₄S)_(n), wherein n=1 to 4. In some instances, the TCR subunitcomprises a TCR extracellular domain. In some instances, the TCR subunitcomprises a TCR transmembrane domain. In some instances, the TCR subunitcomprises a TCR intracellular domain. In some instances, the TCR subunitcomprises (i) a TCR extracellular domain, (ii) a TCR transmembranedomain, and (iii) a TCR intracellular domain, wherein at least two of(i), (ii), and (iii) are from the same TCR subunit. In some instances,the TCR subunit comprises a TCR intracellular domain comprising astimulatory domain selected from an intracellular signaling domain ofCD3 epsilon, CD3 gamma or CD3 delta, or an amino acid sequence having atleast one, two or three modifications thereto. In some instances, theTCR subunit comprises an intracellular domain comprising a stimulatorydomain selected from a functional signaling domain of 4-1BB and/or afunctional signaling domain of CD3 zeta, or an amino acid sequencehaving at least one modification thereto. In some instances, the humanor humanized antibody domain comprises an antibody fragment. In someinstances, the human or humanized antibody domain comprises a scFv or aV_(H) domain. In some instances, the isolated nucleic acid moleculeencodes (i) a light chain (LC) CDR1, LC CDR2 and LC CDR3 of ananti-mesothelin light chain binding domain amino acid sequence with70-100% sequence identity to a light chain (LC) CDR1, LC CDR2 and LCCDR3 of an anti-mesothelin light chain binding domain provided herein,repectively, and/or (ii) a heavy chain (HC) CDR1, HC CDR2 and HC CDR3 ofan anti-mesothelin heavy chain binding domain amino acid sequence with70-100% sequence identity to a heavy chain (HC) CDR1, HC CDR2 and HCCDR3 of an anti-mesothelin heavy chain binding domain provided herein,respectively. In some instances, the isolated nucleic acid moleculeencodes a light chain variable region, wherein the light chain variableregion comprises an amino acid sequence having at least one but not morethan 30 modifications of a light chain variable region amino acidsequence of a light chain variable region provided herein, or a sequencewith 95-99% identity to a light chain variable region amino acidsequence of a light chain variable region provided herein. In someinstances, the isolated nucleic acid molecule encodes a heavy chainvariable region, wherein the heavy chain variable region comprises anamino acid sequence having at least one but not more than 30modifications of a heavy chain variable region amino acid sequence of aheavy chain variable region provided herein, or a sequence with 95-99%identity to a heavy chain variable region amino acid sequence of a heavychain variable region provided herein. In some instances, the TFPincludes an extracellular domain of a TCR subunit that comprises anextracellular domain or portion thereof of a protein selected from thegroup consisting of a TCR alpha chain, a TCR beta chain, a CD3 epsilonTCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit,functional fragments thereof, and amino acid sequences thereof having atleast one but not more than 20 modifications. In some instances, theencoded TFP includes a transmembrane domain that comprises atransmembrane domain of a protein selected from the group consisting ofa TCR alpha chain, a TCR beta chain, a CD3 epsilon TCR subunit, a CD3gamma TCR subunit, a CD3 delta TCR subunit, functional fragmentsthereof, and amino acid sequences thereof having at least one but notmore than 20 modifications. In some instances, the encoded TFP includesa transmembrane domain that comprises a transmembrane domain of aprotein selected from the group consisting of a TCR alpha chain, a TCRbeta chain, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCRsubunit, a CD3 delta TCR subunit, CD45, CD2, CD4, CD5, CD8, CD9, CD16,CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154,functional fragments thereof, and amino acid sequences thereof having atleast one but not more than 20 modifications. In some instances, theisolated nucleic acid molecule further comprises a sequence encoding acostimulatory domain. In some instances, the costimulatory domain is afunctional signaling domain obtained from a protein selected from thegroup consisting of DAP10, DAP12, CD30, LIGHT, OX40, CD2, CD27, CD28,CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), and 4-1BB (CD137), andamino acid sequences thereof having at least one but not more than 20modifications thereto. In some instances, the isolated nucleic acidmolecule further comprises a leader sequence. In some instances, theisolated nucleic acid molecule is mRNA.

In some instances, the TFP includes an immunoreceptor tyrosine-basedactivation motif (ITAM) of a TCR subunit that comprises an ITAM orportion thereof of a protein selected from the group consisting of CD3zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, CD3delta TCR subunit, TCR zeta chain, Fc epsilon receptor 1 chain, Fcepsilon receptor 2 chain, Fc gamma receptor 1 chain, Fc gamma receptor2a chain, Fc gamma receptor 2b1 chain, Fc gamma receptor 2b2 chain, Fcgamma receptor 3a chain, Fc gamma receptor 3b chain, Fc beta receptor 1chain, TYROBP (DAP12), CD5, CD16a, CD16b, CD22, CD23, CD32, CD64, CD79a,CD79b, CD89, CD278, CD66d, functional fragments thereof, and amino acidsequences thereof having at least one but not more than 20 modificationsthereto. In some instances, the ITAM replaces an ITAM of CD3 gamma, CD3delta, or CD3 epsilon. In some instances, the ITAM is selected from thegroup consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3gamma TCR subunit, and CD3 delta TCR subunit and replaces a differenctITAM selected from the group consisting of CD3 zeta TCR subunit, CD3epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit.

In some instances, the nucleic acid comprises a nucleotide analog. Insome instances, the nucleotide analog is selected from the groupconsisting of 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE),2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl(2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE),2′-O-dimethylaminopropyl (2′-O-DMAP), T-O-dimethylaminoethyloxyethyl(2′-O-DMAEOE), 2′-O—N-methylacetamido (2′-O-NMA) modified, a lockednucleic acid (LNA), an ethylene nucleic acid (ENA), a peptide nucleicacid (PNA), a 1′,5′-anhydrohexitol nucleic acid (HNA), a morpholino, amethylphosphonate nucleotide, a thiolphosphonate nucleotide, and a2′-fluoro N3-P5′-phosphoramidite.

In one aspect, provided herein is an isolated polypeptide moleculeencoded by a nucleic acid molecule provided herein.

In one aspect, provided herein is an isolated TFP molecule comprising ahuman or humanized anti-mesothelin binding domain, a TCR extracellulardomain, a transmembrane domain, and an intracellular domain.

In one aspect, provided herein is an isolated TFP molecule comprising ahuman or humanized anti-mesothelin binding domain, a TCR extracellulardomain, a transmembrane domain, and an intracellular signaling domain,wherein the TFP molecule is capable of functionally interacting with anendogenous TCR complex and/or at least one endogenous TCR polypeptide.

In some embodiments, the human or humanized antibody domain comprisingan antigen binding domain that is an anti-mesothelin binding domainencoded by the nucliec acid, or an antibody comprising theanti-mesothelin binding domain, or a cell expressing the anti-mesothelinbinding domain encoded by the nucliec acid has an affinity value of atmost about 200 nM, 100 nM, 75 nM, a 50 nM, 25 nM, 20 nM, 15 nM, 14 nM,13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03nM, 0.02 nM, or 0.01 nM; and/or at least about 100 nM, 75 nM, a 50 nM,25 nM, 20 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM,0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM, or 0.01 nM; and or about 200 nM,100 nM, 75 nM, a 50 nM, 25 nM, 20 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM,10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM,0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM, or 0.01nM.

In one aspect, provided herein is an isolated TFP molecule comprising ahuman or humanized anti-mesothelin binding domain, a TCR extracellulardomain, a transmembrane domain, and an intracellular signaling domain,wherein the TFP molecule is capable of functionally integrating into anendogenous TCR complex

In some instances, the isolated TFP molecule comprises an antibody orantibody fragment comprising a human or humanized anti-mesothelinbinding domain, a TCR extracellular domain, a transmembrane domain, andan intracellular domain. In some instances, the anti-mesothelin bindingdomain is a scFv, a V_(H) domain, or a camelid V_(HH) domain. In someinstances, the anti-mesothelin binding domain comprises a heavy chainwith 95-100% identity to an amino acid sequence of a heavy chainprovided herein, a functional fragment thereof, or an amino acidsequence thereof having at least one but not more than 30 modifications.In some instances, the anti-mesothelin binding domain comprises a lightchain with 95-100% identity to an amino acid sequence of a light chainprovided herein, a functional fragment thereof, or an amino acidsequence thereof having at least one but not more than 30 modifications.In some instances, the isolated TFP molecule comprises a TCRextracellular domain that comprises an extracellular domain or portionthereof of a protein selected from the group consisting of a TCR alphachain, a TCR beta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCRsubunit, a CD3 delta TCR subunit, functional fragments thereof, andamino acid sequences thereof having at least one but not more than 20modifications. In some instances, the anti-mesothelin binding domain isconnected to the TCR extracellular domain by a linker sequence. In someinstances, the linker region comprises (G₄S)_(n), wherein n=1 to 4.

In some instances, the isolated TFP molecule further comprises asequence encoding a costimulatory domain. In some instances, theisolated TFP molecule further comprises a sequence encoding anintracellular signaling domain. In some instances, the isolated TFPmolecule further comprises a leader sequence.

In one aspect, provided herein is a vector comprising a nucleic acidmolecule encoding a TFP provided herein. In some instances, the vectoris selected from the group consisting of a DNA, a RNA, a plasmid, alentivirus vector, adenoviral vector, a Rous sarcoma viral (RSV) vector,or a retrovirus vector. In some instances, the vector further comprisesa promoter. In some instances, the vector is an in vitro transcribedvector. In some instances, a nucleic acid sequence in the vector furthercomprises a poly(A) tail. In some instances, a nucleic acid sequence inthe vector further comprises a 3′UTR.

In one aspect, provided herein is a cell comprising a vector providedherein. In some instances, the cell is a human T-cell. In someinstances, the T-cell is a CD8+ or CD4+ T-cell. In some instances, the Tcell is a gamma delta T cell. In some instances, the T cell is an NK-Tcell. In some instances, the cell further comprises a nucleic acidencoding an inhibitory molecule that comprises a first polypeptide thatcomprises at least a portion of an inhibitory molecule, associated witha second polypeptide that comprises a positive signal from anintracellular signaling domain. In some instances, the inhibitorymolecule comprise first polypeptide that comprises at least a portion ofPD1 and a second polypeptide comprising a costimulatory domain andprimary signaling domain.

In one aspect, provided herein is a human CD8+ or CD4+ T-cell comprisingat least two TFP molecules, the TFP molecules comprising a human orhumanized anti-mesothelin binding domain, a TCR extracellular domain, atransmembrane domain, and an intracellular domain, wherein the TFPmolecule is capable of functionally interacting with an endogenous TCRcomplex and/or at least one endogenous TCR polypeptide in, at and/or onthe surface of the human CD8+ or CD4+ T-cell.

In one aspect, provided herein is a protein complex comprising: a TFPmolecule comprising a human or humanized anti-mesothelin binding domain,a TCR extracellular domain, a transmembrane domain, and an intracellulardomain; and at least one endogenous TCR complex.

In some instances, the TCR comprises an extracellular domain or portionthereof of a protein selected from the group consisting of TCR alphachain, a TCR beta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCRsubunit, and a CD3 delta TCR subunit. In some instances, theanti-mesothelin binding domain is connected to the TCR extracellulardomain by a linker sequence. In some instances, the linker regioncomprises (G₄S)_(n), wherein n=1 to 4.

In one aspect, provided herein is a human CD8+ or CD4+ T-cell comprisingat least two different TFP proteins per a protein complex providedherein.

In one aspect, provided herein is a method of making a cell comprisingtransducing a T-cell with a vector provided herein.

In one aspect, provided herein is a method of generating a population ofRNA-engineered cells comprising introducing an in vitro transcribed RNAor synthetic RNA into a cell, where the RNA comprises a nucleic acidencoding a TFP molecule provided herein.

In one aspect, provided herein is a method of providing an anti-tumorimmunity in a mammal comprising administering to the mammal an effectiveamount of a cell expressing a TFP molecule provided herein, orexpressing a polypeptide molecule provided herein.

In some instances, the cell is an autologous T-cell. In some instances,the cell is an allogeneic T-cell. In some instances, the mammal is ahuman.

In one aspect, provided herein is a method of treating a mammal having adisease associated with expression of mesothelin comprisingadministering to the mammal an effective amount of a TFP moleculeprovided herein, a cell provided herein, or a polypeptide moleculeprovided herein. In some instances, the disease associated withmesothelin expression is selected from the group consisting of aproliferative disease, a cancer, a malignancy, and a non-cancer relatedindication associated with expression of mesothelin. In some instances,the disease is a cancer selected from the group consisting ofmesothelioma, renal cell carcinoma, stomach cancer, breast cancer, lungcancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer,brain cancer, liver cancer, pancreatic cancer, thyroid cancer, bladdercancer, ureter cancer, kidney cancer, endometrial cancer, esophogealcancer, gastric cancer, thymic carcinoma, cholangiocarcinoma and stomachcancer.

In some instances, the disease is cancer. In some instances, the diseaseis selected from the group consisting of mesothelioma, papillary serousovarian adenocarcinoma, clear cell ovarian carcinoma, mixed Mullerianovarian carcinoma, endometroid mucinous ovarian carcinoma, malignangpleural disease, pancreatic adenocarcinoma, ductal pancreaticadenocarcinoma, uterine serous carcinoma, lung adenocarcinoma,extrahepatic bile duct carcinoma, gastric adenocarcinoma, esophagealadenocarcinoma, colorectal adenocarcinoma, breast adenocarcinoma, adisease associated with mesothelin expression, a disease associated withmesothelin expression, non-mucinous ovarian carcinoma, invasive ductaladenocarcinoma, pulmonary adenocarcinoma, gastric/esophagealadenocarcinoma, colorectal adenocarcinoma, leukemia, pediatric acutemyeloid leukemia, invasive intraductal papillary mucinous neoplasm(IPMN), endometrial adenocarcinoma, stomach/esophagus adenocarcinoma,pulmonary adenocarcinoma, breast adenocarcinoma, and combinationsthereof.

In some instances, the cells expressing a TFP molecule are administeredin combination with an agent that increases the efficacy of a cellexpressing a TFP molecule. In some instances, less cytokines arereleased in the mammal compared a mammal administered an effectiveamount of a T-cell expressing an anti-mesothelin chimeric antigenreceptor (CAR). In some instances, the cells expressing a TFP moleculeare administered in combination with an agent that ameliorates one ormore side effects associated with administration of a cell expressing aTFP molecule. In some instances, the cells expressing a TFP molecule areadministered in combination with an agent that treats the diseaseassociated with mesothelin.

In one aspect, an isolated nucleic acid molecule provided herein, anisolated polypeptide molecule provided herein, an isolated TFP providedherein, a complex provided herein, a vector provided herein, or a cellprovided herein, is for use as a medicament.

In one aspect, provided herein is a method of treating a mammal having adisease associated with expression of mesothelin comprisingadministering to the mammal an effective amount of a TFP moleculeprovided herein, a cell provided herein, or a polypeptide moleculeprovided herein, wherein less cytokines are released in the mammalcompared a mammal administered an effective amount of a T-cellexpressing an anti-mesothelin chimeric antigen receptor (CAR).

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 is a schematic illustration demonstrating the use of T-cellreceptor fusion polypeptides (TFPs) of the invention. An exemplary TFPcontains an anti-mesothelin scFv and a full-length CD3 epsilonpolypeptide fused via a (G₄S)₃ linker sequence. When produced by orintroduced into a T-cell, the TFP associates with other polypeptides ofthe endogenous T-cell receptor (TCR) (shown to include two CD3 epsilonpolypeptides, one CD3 gamma polypeptide, one CD3 delta polypeptide, twoCD3 zeta polypeptides, one TCR alpha subunit and one TCR beta subunit,where the horizontal grey segment represents the plasma membrane) toform a reprogrammed TCR in which one or both of the endogenous CD3epsilon polypeptides are substituted by the TFP.

FIGS. 2A-D represents schematic illustrations demonstrating exemplaryvariations of reprogrammed T-cell receptor fusion polypeptides (TFPs) ofthe invention. FIG. 2A illustration denoted scFv:TCR-Va illustrates anexemplary reprogrammed TCR containing a TFP that contains ananti-mesothelin scFv and a full-length TCR-Vα polypeptide fused via a(G₄S)₃ linker sequence. FIG. 2B illustration denoted scFv:TCR-Vα:TCR-Vβillustrates an exemplary reprogrammed TCR that contain multiple TFPsincluding i) an anti-mesothelin scFv and a full-length TCR-Vαpolypeptide fused via a (G₄S)₃ linker sequence and ii) ananti-mesothelin scFv and a full-length TCR-VP polypeptide fused via a(G₄S)₃ linker sequence. FIG. 2C illustration denoted scFv:ATCR-Vα:CD3εillustrates an exemplary reprogrammed TCR that contains multiple TFPsincluding i) an anti-mesothelin scFv and a truncated (A) TCR polypeptidefused via a (G₄S)₃ linker sequence and ii) an anti-mesothelin scFv and afull-length CD3 epsilon polypeptide fused via a (G₄S)₃ linker sequence.The truncated (A) TCR polypeptide is truncated by the deletion of theVa. FIG. 2D illustration denoted scFv:ATCR-Vα:ATCR-Vβ illustrates anexemplary reprogrammed TCR that contains multiple TFPs including i) ananti-mesothelin scFv and a truncated (A) TCR Vα polypeptide fused via a(G₄S)₃ linker sequence and ii) an anti-mesothelin scFv and a truncated(A) TCR VP polypeptide fused via a (G₄S)₃ linker sequence. The truncated(A) TCR polypeptide is truncated by the deletion of the Vβ.

FIG. 3 is a schematic illustration demonstrating the use of T-cellreceptor fusion polypeptides (TFPs) of the invention. An exemplary TFPcontains an anti-mesothelin V_(H) domain and a full-length CD3 epsilonpolypeptide fused via a (G₄S)₃ linker sequence. When produced by aT-cell or introduced into a T-cell, the TFP associates with otherpolypeptides of the endogenous T-cell receptor (TCR) (shown to includetwo CD3 epsilon polypeptides, one CD3 gamma polypeptide, one CD3 deltapolypeptide, two CD3 zeta polypeptides, one TCR alpha subunit and oneTCR beta subunit, where the horizontal grey segment represents theplasma membrane) to form a reprogrammed TCR in which one or both of theendogenous CD3 epsilon polypeptides are substituted by the TFP.

FIG. 4 is a series of schematic illustrations demonstrating DNAconstructs encoding various TFPs.

FIG. 5A depicts exemplary surface expression analysis of TFPs onactivated PBMC cells and shows CD3⁺ cells (anti-CD3 APC, gate) activatedwith MSLN TFPs and stained for CD8 (anti-CD8 APCCy7, y-axes) andmesothelin (“MSLN”) (Zenon® R-Phycoerythrin-labeled hMSLN IgG, x-axes).Shown from left to right are cells that were either non-transduced ortransduced with anti-MSLN-CD3ε TFP, anti-MSLN-CD28ζ CAR, andanti-MSLN-41BBζ CAR constructs.

FIG. 5B depicts exemplary surface expression analysis of TFPs onactivated PBMC cells and shows cells activated with in-house singledomain TFPs and stained for MSLN Fc and and analyzed for GFP. The toprow shows (from left to right) non-transduced cells, and cellstransduced with a control anti-MSLN-CD3ε TFP (“SS1”). Rows 2-4 show theanti-MSLN binders SD1, SD4, and SD6, respectively, in cells transducedwith GFP-tagged (from left to right) CD3ε TFP, CD3γTFP, TCRβ TFP, andCD28ζ CAR constructs.

FIG. 6A is an exemplary graph depicting killing of mesothelin(MSLN)-positive HeLa (cervical adenocarcinoma, ATCC® CCL-2™) targetcells by anti-MSLN-TFP constructs overtime. Activated PBMCs wereuntreated (trace #1), non-transduced (trace #2), or transduced withempty vector (trace #3), anti-MSLN-CD3ε TFP (trace #4), anti-MSLN-CD28ζCAR, or anti-MSLN-41BBζ CAR and expanded for 8 days prior to incubationwith 1×104 MSLN-positive HeLa target cells.

FIG. 6B is an exemplary graph depicting killing of MSLN-negative HeLa(cervical adenocarcinoma, ATCC® CCL-2™) target cells by anti-MSLN-TFPconstructs overtime. Activated PBMCs were untreated (trace #1),non-transduced (trace #2), or transduced with empty vector (trace #3),anti-MSLN-CD3ε TFP (trace #4), anti-MSLN-CD28ζ CAR, or anti-MSLN-41BBζCAR and expanded for 8 days prior to incubation with 1×104 MSLN-positiveHeLa target cells.

FIG. 6C shows killing of MSLN-positive cells in a high MSLN-expressingcell line (HeLa cells) using T cells from two different human donors(top and bottom). Shown are the cell killing traces for TFP T cells withthe in-house anti-MSLN binders SD1 (FIG. 7A), SD4 (middle), and SD6(right). Activated PBMCs were nontransduced (trace #1), ortransducedwith CD3ε TFP (trace #2), CD3γ TFP (trace #3), TCRβ TFP (trace #4), orCD28ζ CAR. The normalized cell index, indicative of cytotoxicity, wasdetermined in a real time cell analyzer (RTCA) assay.

FIGS. 7A-C are a series of graphs showing binding activity of anti-MSLNCAR T cells and TFP T cells against a target cell line expressing highlevels of mesothelin (HeLa-Luc(^(MSLN high))) Shown are the % of cellskilled in samples with no T cells (“target only”), empty vectortransduced (“NT”), anti-MSLN (positive control), or anti-mesothelin TFPT cells with in-house anti-mesothelin binders SD1 (FIG. 7A), SD4 (FIG.7B), and SD6 (FIG. 7C), each in each in the format of CD3ε TFP, CD3γTFP, TCR TFP, and CD28ζ CAR. In each graph, black bars represent a 1:1ratio of T cells to target cells, and gray bars represent a 1:5 ratio ofT cells to target cells. Similar results were seen for a second T celldonor.

FIGS. 8A-D are a series of graphs showing the activity of anti-MSLN CART cells and TFP T cells against a target cell line expressing low levelsof mesothelin (PC3-MSLN(^(−/low))). Shown are the % of cells killed insamples with no T cells (“target only”), empty vector transduced (“NT”),anti-MSLN (positive control, “SS1”), or in-house anti-mesothelinconstructs SD1, SD4, and SD6 in the TFP formats CD3ε (FIG. 8A), CD3γ(FIG. 8B), TCRβ (FIG. 8C), and CD28ζ CAR (FIG. 8D). In each graph, blackbars represent a 1:1 ratio of T cells to target cells, and gray barsrepresent a 1:5 ratio of T cells to target cells. Similar results wereseen for a second T cell donor.

FIGS. 9A-D show the results of FACS analysis demonstrating activation ofT-cells expressing anti-MSLN CAR and TFP constructs when co-culturedwith MSLN+ cells. As shown in FIG. 9A, from left to right, T cells wereeither non-transduced, transduced with empty vector, transduced withanti-MSLN-CD3ε TFP, anti-MSLN-28ζ CAR, or anti-MSLN-41BBζ CAR. Cellsco-cultured with MSLN− cells are shown in the top row, and thoseco-cultured with MSLN+ target cells are shown in the bottom row. Thecells were then stained with antibodies specific for the surfaceactivation markers CD69 and CD25 or the cytolylic granule componentgranzyme B (GrB). The numbers of cells stained with anti-CD69 correspondto the x-axes and those stained with anti-CD25 correspond to the y-axes.As shown, T-cells expressing anti-mesothelin CAR and TFP constructs wereactivated by culturing with MSLN+ cells, as demonstrated by elevatedlevels of CD69 and CD25 expression, relative to co-culturing with MSLN−cells (FIG. 9B). The percentage of CD25+ cells for each construct inMSLN-(white bars) and MSLN+(black bars) cells is shown. A similarexperiment was done using K562 MSLN− cells (circles) and K562-MSLN+cells (squares) in either non-transduced T cells or T cells transducedwith anti-MSLN positive control binders (“510-SS1-CD3ε) (FIG. 9C). Datarepresent the sum of CD25+, CD69+, and CD25+/CD69+ cells. In FIG. 9D,data are shown for the in-house anti-MSLN binders SD (squares), SD4(circles), and SD6 (triangles) in K562 MSLN-target cells (left panel)and K562 MSLN+ cells (right panel) combined with donor T cells havingTFP formats CD3ε, CD3γ, TCRβ, and CD28 ζ CAR. Similar results were seenusing cells from a second T cell donor.

FIGS. 10A-B show the results of FACS analysis demonstrating activationof T-cells expressing anti-MSLN CAR and TFP constructs when co-culturedwith MSLN+ cells. Cells were stained for surface antigens with anti-CD3APC (gate) and anti-CD8 APCcy7(y-axes) priorto fixation,permealbilzation and staining with anti-Granzyme B Alexafluor700(x-axes). As shown in FIG. 10A, from left to right, T cells were eithernon-transduced, transduced with empty vector, transduced withAnti-MSLN-CD3ε TFP, anti-MSLN-28ζ CAR, or anti-MSLN-41BBζ CAR. Cellsco-cultured with MSLN− cells are shown in the top row, and thoseco-cultured with MSLN+ target cells are shown in the bottom row. CD8T-cells expressing anti-mesothelin CAR and TFP constructs were activatedby culturing with MSLN+ cells, as shown by elevated levels ofintracellular GrB, compared to co-culturing with MSLN− cells (FIG. 10B).The percentage of granzyme B (“GrB+”) cells for each construct, uponcoculture with either MSLN-(white bars) or MSLN+(black bars) cells, isshown.

FIGS. 11A-B show the results of ELISA analysis of cytokine production inactivated T-cells expressing anti-MSLN CAR and TFP constructs whenco-cultured with K562 cells overexpressing MSLN. K562 cellsoverexpressing BCMA were used as negative controls. After 24 hours cellswere analyzed for IFN-γ (FIG. 11A) and IL-2 (FIG. 11B) expression byELISA. In each FIG., from left to right, T cells were eithernon-transduced, transduced with empty vector, transduced withAnti-MSLN-CD3ε TFP, anti-MSLN-28ζ CAR, or anti-MSLN-41BBζ CAR. Cellsco-cultured with MSLN− cells are represented by white bars, and thoseco-cultured with MSLN+ target cells are represented by black bars.

FIGS. 12A-D are a series of graphs showing the efficacy of MSLN-specificsdAb TFP T cells in vivo in a mesothelioma xenograft mouse model. Micewere inoculated with luciferase-labeled MSTO-211H-FL-MSLN-Luc at 1×10⁶cells per mouse and tumors were grown until the tumor volume wasapproximately 300 mm³, 1×10⁷ T cells were injected intravenously intoeach animal. FIG. 12A shows the tumor volume after injection with Tcells including, from left to right, a no T cell control, SD CD3ε-TFP,and SD4 CD3ε-TFP. FIG. 12B shows CD3γ-TFPs with SD1 and SD4 binders andSD1 CD28ζ CAR.

FIGS. 12C-D shows results from surviving mice from FIGS. 12A-B that werere-challenged with tumor cells in order to determine whether the micewould maintain their anti-MSLN immunity without a second T cellinjection. Mice that had been administered SD1 CD3ε-TFP T cells (FIG.12C) and SD1 CD3γ-TFP T cells (FIG. 12D) and had previously clearedtheir tumors, were re-inoculated with either MSLN+(MSTO) or MSLN-(Raji)tumor cell lines. Tumor volume was measured and shown on the x-axis.

FIGS. 13A-L shows production and functional analysis of MSLN-TFP T cellsfrom ovarian cancer patients. FIG. 13A is a schematic diagram of theexperimental design. FIGS. 13B-C show in vitro killing of MSTO-MSLN-Luctumor cells by patients' SD1 ε-TFP T cells. MSTO-MSLN-Luc tumor cells(target cells) were confirmed for mesothelin expression (FIG. 13B); SD1ε-TFP T cells (effector cells) and matching non-transduced control wereadded at E-to-T (effector to target) ratios 5-to-1, 1-to-1, or 1-to-5for 24 hours. The luminescence of target cells was measured relativeluminescence unit (RLU) by SpectraMax® M5 plate reader (Moleculardevices). Each line in the figure represents the average of 3 replicates(FIG. 13C). FIGS. 13D-L show measurement of the cytokine profile of SD1F-TFP T cells from ovarian cancer patients, including IFNγ (FIG. 13D),GM-CSF (FIG. 13E), Granzyme A (FIG. 13F), Granzyme B (FIG. 13G), IL-2(FIG. 13H), MIP-1α (FIG. 13I), MIP-10 (FIG. 13J), TNFα (FIG. 13K), andperforin (FIG. 13L). MSTO-MSLN-Luc tumor cells (target cells) wereplated at 10000 cells/well in 96 flat bottom plate. SD1 ε-TFP T cells(effector cells) and a matching non-transduced control were added at1-to-1 ratio for 24 hours. Cell supernatants were collected andcytokines were measured using a Luminex® assay.

FIGS. 14A-E shows the in vivo efficacy of patient-derived SD1 CD3ε-TFP Tcells in MSLN-high xenograft tumor mouse model. MSTO-211H-FL MSLN-Luccells were inoculated at 1×10⁶ cells per mouse subcutaneously. Ten daysafter tumor injection (tumor volume ˜200-300 mm³), 5×10⁶ T cells wereinjected intravenously into each animal. Each line in the figurerepresents single animal. Data are shown for T cells from ND12 (FIG.14A), Patient 1 (FIG. 14B), Patient 2 (FIG. 14C), Patient 3 (FIG. 14D),and Patient 4 (FIG. 14E). Circles indicate tumor size in mice inoculatedwith untransduced T cells; squares indicate those inoculated with TFP Tcells.

DETAILED DESCRIPTION

In one aspect, described herein are isolated nucleic acid moleculesencoding a T-cell Receptor (TCR) fusion protein (TFP) that comprise aTCR subunit and a human or humanized antibody domain comprising ananti-mesothelin binding domain. In some embodiments, the TCR subunitcomprises a TCR extracellular domain. In other embodiments, the TCRsubunit comprises a TCR transmembrane domain. In yet other embodiments,the TCR subunit comprises a TCR intracellular domain. In furtherembodiments, the TCR subunit comprises (i) a TCR extracellular domain,(ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain,wherein at least two of (i), (ii), and (iii) are from the same TCRsubunit. In yet further embodiments, the TCR subunit comprises a TCRintracellular domain comprising a stimulatory domain selected from anintracellular signaling domain of CD3 epsilon, CD3 gamma or CD3 delta,or an amino acid sequence having at least one, two or threemodifications thereto. In yet further embodiments, the TCR subunitcomprises an intracellular domain comprising a stimulatory domainselected from a functional signaling domain of 4-1BB and/or a functionalsignaling domain of CD3 zeta, or an amino acid sequence having at leastone, two or three modifications thereto.

In some embodiments, the isolated nucleic acid molecules comprise (i) alight chain (LC) CDR1, LC CDR2 and LC CDR3 of any anti-mesothelin lightchain binding domain amino acid sequence provided herein, and/or (ii) aheavy chain (HC) CDR1, HC CDR2 and HC CDR3 of any anti-mesothelin heavychain binding domain amino acid sequence provided herein.

In some embodiments, the light chain variable region comprises an aminoacid sequence having at least one, two or three modifications but notmore than 30, 20 or 10 modifications of an amino acid sequence of alight chain variable region provided herein, or a sequence with 95-99%identity to an amino acid sequence provided herein. In otherembodiments, the heavy chain variable region comprises an amino acidsequence having at least one, two or three modifications but not morethan 30, 20 or 10 modifications of an amino acid sequence of a heavychain variable region provided herein, or a sequence with 95-99%identity to an amino acid sequence provided herein.

In some embodiments, the TFP includes an extracellular domain of a TCRsubunit that comprises an extracellular domain or portion thereof of aprotein selected from the group consisting of the alpha or beta chain ofthe T-cell receptor, CD3 delta, CD3 epsilon, or CD3 gamma, or afunctional fragment thereof, or an amino acid sequence having at leastone, two or three modifications but not more than 20, 10 or 5modifications thereto. In other embodiments, the encoded TFP includes atransmembrane domain that comprises a transmembrane domain of a proteinselected from the group consisting of the alpha, beta chain of the TCRor TCR subunits CD3 epsilon, CD3 gamma and CD3 delta, or a functionalfragment thereof, or an amino acid sequence having at least one, two orthree modifications but not more than 20, 10 or 5 modifications thereto.

In some embodiments, the encoded TFP includes a transmembrane domainthat comprises a transmembrane domain of a protein selected from thegroup consisting of the alpha, beta or zeta chain of the TCR or CD3epsilon, CD3 gamma and CD3 delta CD45, CD2, CD4, CD5, CD8, CD9, CD16,CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, or afunctional fragment thereof, or an amino acid sequence having at leastone, two or three modifications but not more than 20, 10 or 5modifications thereto.

In some embodiments, the encoded anti-mesothelin binding domain isconnected to the TCR extracellular domain by a linker sequence. In someinstances, the encoded linker sequence comprises (G₄S)_(n), wherein n=1to 4. In some instances, the encoded linker sequence comprises a longlinker (LL) sequence. In some instances, the encoded long linkersequence comprises (G₄S)_(n), wherein n=2 to 4. In some instances, theencoded linker sequence comprises a short linker (SL) sequence. In someinstances, the encoded short linker sequence comprises (G₄S)_(n),wherein n=1 to 3.

In some embodiments, the isolated nucleic acid molecules furthercomprise a sequence encoding a costimulatory domain. In some instances,the costimulatory domain is a functional signaling domain obtained froma protein selected from the group consisting of DAP10, DAP12, CD30,LIGHT, OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS(CD278), and 4-1BB (CD137), or an amino acid sequence having at leastone, two or three modifications but not more than 20, 10 or 5modifications thereto.

In some embodiments, the isolated nucleic acid molecules furthercomprise a leader sequence.

Also provided herein are isolated polypeptide molecules encoded by anyof the previously described nucleic acid molecules.

Also provided herein in another aspect, are isolated T-cell receptorfusion protein (TFP) molecules that comprise a human or humanizedanti-mesothelin binding domain, a TCR extracellular domain, atransmembrane domain, and an intracellular domain. In some embodiments,the isolated TFP molecules comprises an antibody or antibody fragmentcomprising a human or humanized anti-mesothelin binding domain, a TCRextracellular domain, a transmembrane domain, and an intracellulardomain.

In some embodiments, the human or humanized antibody domain comprises anantibody fragment. In some embodiments, the human or humanized antibodydomain comprises a scFv or a V_(H) domain.

In some embodiments, the anti-mesothelin binding domain is a scFv or aV_(H) domain. In other embodiments, the anti-mesothelin binding domaincomprises a light chain and a heavy chain of an amino acid sequenceprovided herein, or a functional fragment thereof, or an amino acidsequence having at least one, two or three modifications but not morethan 30, 20 or 10 modifications of an amino acid sequence of a lightchain variable region provided herein, or a sequence with 95-99%identity with an amino acid sequence provided herein.

In some embodiments, the isolated TFP molecules comprise a TCRextracellular domain that comprises an extracellular domain or portionthereof of a protein selected from the group consisting of the alpha orbeta chain of the T-cell receptor, CD3 delta, CD3 epsilon, or CD3 gamma,or an amino acid sequence having at least one, two or threemodifications but not more than 20, 10 or 5 modifications thereto.

In some embodiments, the anti-mesothelin binding domain is connected tothe TCR extracellular domain by a linker sequence. In some instances,the linker region comprises (G₄S)_(n), wherein n=1 to 4. In someinstances, the linker sequence comprises a long linker (LL) sequence. Insome instances, the long linker sequence comprises (G₄S)_(n), whereinn=2 to 4. In some instances, the linker sequence comprises a shortlinker (SL) sequence. In some instances, the short linker sequencecomprises (G₄S)_(n), wherein n=1 to 3.

In some embodiments, the isolated TFP molecules further comprise asequence encoding a costimulatory domain. In other embodiments, theisolated TFP molecules further comprise a sequence encoding anintracellular signaling domain. In yet other embodiments, the isolatedTFP molecules further comprise a leader sequence.

Also provided herein are vectors that comprise a nucleic acid moleculeencoding any of the previously described TFP molecules. In someembodiments, the vector is selected from the group consisting of a DNA,a RNA, a plasmid, a lentivirus vector, adenoviral vector, or aretrovirus vector. In some embodiments, the vector further comprises apromoter. In some embodiments, the vector is an in vitro transcribedvector. In some embodiments, a nucleic acid sequence in the vectorfurther comprises a poly(A) tail. In some embodiments, a nucleic acidsequence in the vector further comprises a 3′UTR.

Also provided herein are cells that comprise any of the describedvectors. In some embodiments, the cell is a human T-cell. In someembodiments, the cell is a CD8+ or CD4+ T-cell. In other embodiments,the cells further comprise a nucleic acid encoding an inhibitorymolecule that comprises a first polypeptide that comprises at least aportion of an inhibitory molecule, associated with a second polypeptidethat comprises a positive signal from an intracellular signaling domain.In some instances, the inhibitory molecule comprise first polypeptidethat comprises at least a portion of PD1 and a second polypeptidecomprising a costimulatory domain and primary signaling domain.

In another aspect, provided herein are isolated TFP molecules thatcomprise a human or humanized anti-mesothelin binding domain, a TCRextracellular domain, a transmembrane domain, and an intracellularsignaling domain, wherein the TFP molecule is capable of functionallyinteracting with an endogenous TCR complex and/or at least oneendogenous TCR polypeptide.

In another aspect, provided herein are isolated TFP molecules thatcomprise a human or humanized anti-mesothelin binding domain, a TCRextracellular domain, a transmembrane domain, and an intracellularsignaling domain, wherein the TFP molecule is capable of functionallyintegrating into an endogenous TCR complex.

In another aspect, provided herein are human CD8+ or CD4+ T-cells thatcomprise at least two TFP molecules, the TFP molecules comprising ahuman or humanized anti-mesothelin binding domain, a TCR extracellulardomain, a transmembrane domain, and an intracellular domain, wherein theTFP molecule is capable of functionally interacting with an endogenousTCR complex and/or at least one endogenous TCR polypeptide in, at and/oron the surface of the human CD8+ or CD4+ T-cell.

In another aspect, provided herein are protein complexes that comprisei) a TFP molecule comprising a human or humanized anti-mesothelinbinding domain, a TCR extracellular domain, a transmembrane domain, andan intracellular domain; and ii) at least one endogenous TCR complex.

In some embodiments, the TCR comprises an extracellular domain orportion thereof of a protein selected from the group consisting of thealpha or beta chain of the T-cell receptor, CD3 delta, CD3 epsilon, orCD3 gamma. In some embodiments, the anti-mesothelin binding domain isconnected to the TCR extracellular domain by a linker sequence. In someinstances, the linker region comprises (G₄S)_(n), wherein n=1 to 4. Insome instances, the linker sequence comprises a long linker (LL)sequence. In some instances, the long linker sequence comprises(G₄S)_(n), wherein n=2 to 4. In some instances, the linker sequencecomprises a short linker (SL) sequence. In some instances, the shortlinker sequence comprises (G₄S)_(n), wherein n=1 to 3.

Also provided herein are human CD8+ or CD4+ T-cells that comprise atleast two different TFP proteins per any of the described proteincomplexes.

In another aspect, provided herein is a population of human CD8+ or CD4+T-cells, wherein the T-cells of the population individually orcollectively comprise at least two TFP molecules, the TFP moleculescomprising a human or humanized anti-mesothelin binding domain, a TCRextracellular domain, a transmembrane domain, and an intracellulardomain, wherein the TFP molecule is capable of functionally interactingwith an endogenous TCR complex and/or at least one endogenous TCRpolypeptide in, at and/or on the surface of the human CD8+ or CD4+T-cell.

In another aspect, provided herein is a population of human CD8+ or CD4+T-cells, wherein the T-cells of the population individually orcollectively comprise at least two TFP molecules encoded by an isolatednucleic acid molecule provided herein.

In another aspect, provided herein are methods of making a cellcomprising transducing a T-cell with any of the described vectors.

In another aspect, provided herein are methods of generating apopulation of RNA-engineered cells that comprise introducing an in vitrotranscribed RNA or synthetic RNA into a cell, where the RNA comprises anucleic acid encoding any of the described TFP molecules.

In another aspect, provided herein are methods of providing ananti-tumor immunity in a mammal that comprise administering to themammal an effective amount of a cell expressing any of the described TFPmolecules. In some embodiments, the cell is an autologous T-cell. Insome embodiments, the cell is an allogeneic T-cell. In some embodiments,the mammal is a human.

In another aspect, provided herein are methods of treating a mammalhaving a disease associated with expression of mesothelin that compriseadministering to the mammal an effective amount of the cell ofcomprising any of the described TFP molecules. In some embodiments, thedisease associated with mesothelin expression is selected from aproliferative disease such as a cancer or malignancy or a precancerouscondition such as a pancreatic cancer, an ovarian cancer, a stomachcancer, a lung cancer, or an endometrial cancer, or is a non-cancerrelated indication associated with expression of mesothelin.

In some embodiments, the cells expressing any of the described TFPmolecules are administered in combination with an agent that amelioratesone or more side effects associated with administration of a cellexpressing a TFP molecule. In some embodiments, the cells expressing anyof the described TFP molecules are administered in combination with anagent that treats the disease associated with mesothelin.

Also provided herein are any of the described isolated nucleic acidmolecules, any of the described isolated polypeptide molecules, any ofthe described isolated TFPs, any of the described protein complexes, anyof the described vectors or any of the described cells for use as amedicament

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains.

The term “a” and “an” refers to one or to more than one (i.e., to atleast one) of the grammatical object of the article. By way of example,“an element” means one element or more than one element.

As used herein, “about” can mean plus or minus less than 1 or 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, orgreater than 30 percent, depending upon the situation and known orknowable by one skilled in the art.

As used herein the specification, “subject” or “subjects” or“individuals” may include, but are not limited to, mammals such ashumans or non-human mammals, e.g., domesticated, agricultural or wild,animals, as well as birds, and aquatic animals. “Patients” are subjectssuffering from or at risk of developing a disease, disorder or conditionor otherwise in need of the compositions and methods provided herein.

As used herein, “treating” or “treatment” refers to any indicia ofsuccess in the treatment or amelioration of the disease or condition.Treating can include, for example, reducing, delaying or alleviating theseverity of one or more symptoms of the disease or condition, or it caninclude reducing the frequency with which symptoms of a disease, defect,disorder, or adverse condition, and the like, are experienced by apatient. As used herein, “treat or prevent” is sometimes used herein torefer to a method that results in some level of treatment oramelioration of the disease or condition, and contemplates a range ofresults directed to that end, including but not restricted to preventionof the condition entirely.

As used herein, “preventing” refers to the prevention of the disease orcondition, e.g., tumor formation, in the patient. For example, if anindividual at risk of developing a tumor or other form of cancer istreated with the methods of the present invention and does not laterdevelop the tumor or other form of cancer, then the disease has beenprevented, at least over a period of time, in that individual.

As used herein, a “therapeutically effective amount” is the amount of acomposition or an active component thereof sufficient to provide abeneficial effect or to otherwise reduce a detrimental non-beneficialevent to the individual to whom the composition is administered. By“therapeutically effective dose” herein is meant a dose that producesone or more desired or desirable (e.g., beneficial) effects for which itis administered, such administration occurring one or more times over agiven period of time. The exact dose will depend on the purpose of thetreatment, and will be ascertainable by one skilled in the art usingknown techniques (see, e.g. Lieberman, Pharmaceutical Dosage Forms(vols. 1-3, 1992); Lloyd, The Art, Science and Technology ofPharmaceutical Compounding (1999); and Pickar, Dosage Calculations(1999))

As used herein, a “T-cell receptor (TCR) fusion protein” or “TFP”includes a recombinant polypeptide derived from the various polypeptidescomprising the TCR that is generally capable of i) binding to a surfaceantigen on target cells and ii) interacting with other polypeptidecomponents of the intact TCR complex, typically when co-located in or onthe surface of a T-cell. A “TFP T cell” is a T cell that has beentransduced (e.g., according to the methods disclosed herein) and thatexpresses a TFP, e.g., incorporated into the natural TCR. In someembodiments, the T cell is a CD4+ T cell, a CD8+ T cell, or a CD4+/CD8+T cell. In some embodiments, the TFP T cell is an NK cell. In someembodiments, the TFP T cell is agamma-delta T cell.

As used herein, the term “mesothelin” also known as MSLN or CAK1 antigenor Pre-pro-megakaryocyte-potentiating factor, refers to the protein thatin humans is encoded by the MSLN (or Megakaryocyte-potentiating factor(MPF)) gene. Mesothelin is a 40 kDa protein present on normalmesothelial cells and overexpressed in several human tumors, includingmesothelioma and ovarian and pancreatic adenocarcinoma. The mesothelingene encodes a precursor protein that is processed to yield mesothelinwhich is attached to the cell membrane by a glycophosphatidylinositollinkage and a 31-kDa shed fragment named megakaryocyte-potentiatingfactor (MPF). Mesothelin may be involved in cell adhesion, but itsbiological function is not known. Mesothelin is a tumour differentiationantigen that is normally present on the mesothelial cells lining thepleura, peritoneum and pericardium. Mesothelin is an antigenicdeterminant detectable on mesothelioma cells, ovarian cancer cells,pancreatic adenocarcinoma cell and some squamous cell carcinomas (see,e.g., Kojima et al., J. Biol. Chem. 270:21984-21990(1995) and Onda etal., Clin. Cancer Res. 12:4225-4231(2006)). Mesothelin interacts withCA125/MUC16 (see, e.g., Rump et al., J. Biol. Chem. 279:9190-9198(2004)and Ma et al., J. Biol. Chem. 287:33123-33131(2012)).

The human and murine amino acid and nucleic acid sequences can be foundin a public database, such as GenBank, UniProt and Swiss-Prot. Forexample, the amino acid sequence of human mesothelin can be found asUniProt/Swiss-Prot Accession No. Q13421. The human mesothelinpolypeptide canonical sequence is UniProt Accession No. Q13421 (orQ13421-1):

(SEQ ID NO: 15) MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGET GQEAAPLDGVLANPPNISSLSPRQLLGFPCAEVSGLSTERV RELAVALAQKNVKLSTEQLRCLAHRLSEPPEDLDALPLDLL LFLNPDAFSGPQACTRFFSRITKANVDLLPRGAPERQRLLP AALACWGVRGSLLSEADVRALGGLACDLPGRFVAESAEVLL PRLVSCPGPLDQDQQEAARAALQGGGPPYGPPSTWSVSTMD ALRGLLPVLGQPIIRSIPQGIVAAWRQRSSRDPSWRQPERT ILRPRFRREVEKTACPSGKKAREIDESLIFYKKWELEACVD AALLATQMDRVNAIPFTYEQLDVLKHKLDELYPQGYPESVI QHLGYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMSPQ APRRPLPQVATLIDRFVKGRGQLDKDTLDTLTAFYPGYLCS LSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAF QNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMK LRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQ RQDDLDTLGLGLQGGIPNGYLVLDLSMQEALSGTPCLLGPG  PVLTVLALLLASTLA.

The nucleotide sequence encoding human mesothelin transcript variant 1can be found at Accession No. NM005823. The nucleotide sequence encodinghuman mesothelin transcript variant 2 can be found at Accession No.NM013404. The nucleotide sequence encoding human mesothelin transcriptvariant 3 can be found at Accession No. NM001177355. Mesothelin isexpressed on mesothelioma cells, ovarian cancer cells, pancreaticadenocarcinoma cell and squamous cell carcinomas (see, e.g., Kojima etal., J. Biol. Chem. 270:21984-21990(1995) and Onda et al., Clin. CancerRes. 12:4225-4231(2006)). Other cells that express mesothelin areprovided below in the definition of “disease associated with expressionof mesothelin.” Mesothelin also interacts with CA125/MUC16 (see, e.g.,Rump et al., J. Biol. Chem. 279:9190-9198(2004) and Ma et al., J. Biol.Chem. 287:33123-33131(2012)). In one example, the antigen-bindingportion of TFPs recognizes and binds an epitope within the extracellulardomain of the mesothelin protein as expressed on a normal or malignantmesothelioma cell, ovarian cancer cell, pancreatic adenocarcinoma cell,or squamous cell carcinoma cell.

The term “antibody,” as used herein, refers to a protein, or polypeptidesequences derived from an immunoglobulin molecule, which specificallybinds to an antigen. Antibodies can be intact immunoglobulins ofpolyclonal or monoclonal origin, or fragments thereof and can be derivedfrom natural or from recombinant sources.

The terms “antibody fragment” or “antibody binding domain” refer to atleast one portion of an antibody, or recombinant variants thereof, thatcontains the antigen binding domain, i.e., an antigenic determiningvariable region of an intact antibody, that is sufficient to conferrecognition and specific binding of the antibody fragment to a target,such as an antigen and its defined epitope. Examples of antibodyfragments include, but are not limited to, Fab, Fab′, F(ab′)₂, and Fvfragments, single-chain (sc)Fv (“scFv”) antibody fragments, linearantibodies, single domain antibodies (abbreviated “sdAb”) (either V_(L)or V_(H)), camelid V_(HH) domains, and multi-specific antibodies formedfrom antibody fragments.

The term “scFv” refers to a fusion protein comprising at least oneantibody fragment comprising a variable region of a light chain and atleast one antibody fragment comprising a variable region of a heavychain, wherein the light and heavy chain variable regions arecontiguously linked via a short flexible polypeptide linker, and capableof being expressed as a single polypeptide chain, and wherein the scFvretains the specificity of the intact antibody from which it is derived.

“Heavy chain variable region” or “V_(H)”” (or, in the case of singledomain antibodies, e.g., nanobodies, “V_(HH)”) with regard to anantibody refers to the fragment of the heavy chain that contains threeCDRs interposed between flanking stretches known as framework regions,these framework regions are generally more highly conserved than theCDRs and form a scaffold to support the CDRs.

Unless specified, as used herein a scFv may have the V_(L) and V_(H)variable regions in either order, e.g., with respect to the N-terminaland C-terminal ends of the polypeptide, the scFv may compriseV_(L)-linker-V_(H) or may comprise V_(H)-linker-V_(L).

The portion of the TFP composition of the invention comprising anantibody or antibody fragment thereof may exist in a variety of formswhere the antigen binding domain is expressed as part of a contiguouspolypeptide chain including, for example, a single domain antibodyfragment (sdAb) or heavy chain antibodies HCAb, a single chain antibody(scFv) derived from a murine, humanized or human antibody (Harlow etal., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, N.Y.; Harlow et al., 1989, In: Antibodies: ALaboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc.Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science242:423-426). In one aspect, the antigen binding domain of a TFPcomposition of the invention comprises an antibody fragment. In afurther aspect, the TFP comprises an antibody fragment that comprises ascFv or a sdAb.

The term “antibody heavy chain,” refers to the larger of the two typesof polypeptide chains present in antibody molecules in their naturallyoccurring conformations, and which normally determines the class towhich the antibody belongs.

The term “antibody light chain,” refers to the smaller of the two typesof polypeptide chains present in antibody molecules in their naturallyoccurring conformations. Kappa (“·”) and lambda (“·”) light chains referto the two major antibody light chain isotypes.

The term “recombinant antibody” refers to an antibody that is generatedusing recombinant DNA technology, such as, for example, an antibodyexpressed by a bacteriophage or yeast expression system.

The term should also be construed to mean an antibody which has beengenerated by the synthesis of a DNA molecule encoding the antibody andwhich DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using recombinant DNA or amino acid sequencetechnology which is available and well known in the art.

The term “antigen” or “Ag” refers to a molecule that is capable of beingbound specifically by an antibody, or otherwise provokes an immuneresponse. This immune response may involve either antibody production,or the activation of specific immunologically-competent cells, or both.

The skilled artisan will understand that any macromolecule, includingvirtually all proteins or peptides, can serve as an antigen.Furthermore, antigens can be derived from recombinant or genomic DNA. Askilled artisan will understand that any DNA, which comprises anucleotide sequences or a partial nucleotide sequence encoding a proteinthat elicits an immune response therefore encodes an “antigen” as thatterm is used herein. Furthermore, one skilled in the art will understandthat an antigen need not be encoded solely by a full length nucleotidesequence of a gene. It is readily apparent that the present inventionincludes, but is not limited to, the use of partial nucleotide sequencesof more than one gene and that these nucleotide sequences are arrangedin various combinations to encode polypeptides that elicit the desiredimmune response. Moreover, a skilled artisan will understand that anantigen need not be encoded by a “gene” at all. It is readily apparentthat an antigen can be generated synthesized or can be derived from abiological sample, or might be macromolecule besides a polypeptide. Sucha biological sample can include, but is not limited to a tissue sample,a tumor sample, a cell or a fluid with other biological components.

The term “anti-tumor effect” refers to a biological effect which can bemanifested by various means, including but not limited to, e.g., adecrease in tumor volume, a decrease in the number of tumor cells, adecrease in the number of metastases, an increase in life expectancy,decrease in tumor cell proliferation, decrease in tumor cell survival,or amelioration of various physiological symptoms associated with thecancerous condition. An “anti-tumor effect” can also be manifested bythe ability of the peptides, polynucleotides, cells and antibodies ofthe invention in prevention of the occurrence of tumor in the firstplace.

The term “autologous” refers to any material derived from the sameindividual to whom it is later to be re-introduced into the individual.

The term “allogeneic” refers to any material derived from a differentanimal of the same species or different patient as the individual towhom the material is introduced. Two or more individuals are said to beallogeneic to one another when the genes at one or more loci are notidentical. In some aspects, allogeneic material from individuals of thesame species may be sufficiently unlike genetically to interactantigenically.

The term “xenogeneic” refers to a graft derived from an animal of adifferent species.

The term “cancer” refers to a disease characterized by the rapid anduncontrolled growth of aberrant cells. Cancer cells can spread locallyor through the bloodstream and lymphatic system to other parts of thebody. Examples of various cancers are described herein and include butare not limited to, breast cancer, prostate cancer, ovarian cancer,cervical cancer, skin cancer, pancreatic cancer, colorectal cancer,renal cancer, liver cancer, brain cancer, lung cancer, and the like.

The phrase “disease associated with expression of mesothelin” includes,but is not limited to, a disease associated with expression ofmesothelin or condition associated with cells which express mesothelinincluding, e.g., proliferative diseases such as a cancer or malignancyor a precancerous condition In one aspect, the cancer is a mesothelioma.In one aspect, the cancer is a pancreatic cancer. In one aspect, thecancer is an ovarian cancer. In one aspect, the cancer is a stomachcancer. In one aspect, the cancer is a lung cancer. In one aspect, thecancer is an endometrial cancer. Non-cancer related indicationsassociated with expression of mesothelin include, but are not limitedto, e.g., autoimmune disease, (e.g., lupus, rheumatoid arthritis,colitis), inflammatory disorders (allergy and asthma), andtransplantation.

The term “conservative sequence modifications” refers to amino acidmodifications that do not significantly affect or alter the bindingcharacteristics of the antibody or antibody fragment containing theamino acid sequence. Such conservative modifications include amino acidsubstitutions, additions and deletions. Modifications can be introducedinto an antibody or antibody fragment of the invention by standardtechniques known in the art, such as site-directed mutagenesis andPCR-mediated mutagenesis. Conservative amino acid substitutions are onesin which the amino acid residue is replaced with an amino acid residuehaving a similar side chain. Families of amino acid residues havingsimilar side chains have been defined in the art. These families includeamino acids with basic side chains (e.g., lysine, arginine, histidine),acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polarside chains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine,valine, leucine, isoleucine, proline, phenylalanine, methionine),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, one or more amino acid residues within a TFP of theinvention can be replaced with other amino acid residues from the sameside chain family and the altered TFP can be tested using the functionalassays described herein.

The term “stimulation” refers to a primary response induced by bindingof a stimulatory domain or stimulatory molecule (e.g., a TCR/CD3complex) with its cognate ligand thereby mediating a signal transductionevent, such as, but not limited to, signal transduction via the TCR/CD3complex. Stimulation can mediate altered expression of certainmolecules, and/or reorganization of cytoskeletal structures, and thelike.

The term “stimulatory molecule” or “stimulatory domain” refers to amolecule or portion thereof expressed by a T-cell that provides theprimary cytoplasmic signaling sequence(s) that regulate primaryactivation of the TCR complex in a stimulatory way for at least someaspect of the T-cell signaling pathway. In one aspect, the primarysignal is initiated by, for instance, binding of a TCR/CD3 complex withan MHC molecule loaded with peptide, and which leads to mediation of aT-cell response, including, but not limited to, proliferation,activation, differentiation, and the like. A primary cytoplasmicsignaling sequence (also referred to as a “primary signaling domain”)that acts in a stimulatory manner may contain a signaling motif which isknown as immunoreceptor tyrosine-based activation motif or “ITAM”.Examples of an ITAM containing primary cytoplasmic signaling sequencethat is of particular use in the invention includes, but is not limitedto, those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as“ICOS”) and CD66d.

The term “antigen presenting cell” or “APC” refers to an immune systemcell such as an accessory cell (e.g., a B-cell, a dendritic cell, andthe like) that displays a foreign antigen complexed with majorhistocompatibility complexes (MHC's) on its surface. T-cells mayrecognize these complexes using their T-cell receptors (TCRs). APCsprocess antigens and present them to T-cells.

An “intracellular signaling domain,” as the term is used herein, refersto an intracellular portion of a molecule. The intracellular signalingdomain generates a signal that promotes an immune effector function ofthe TFP containing cell, e.g., a TFP-expressing T-cell. Examples ofimmune effector function, e.g., in a TFP-expressing T-cell, includecytolytic activity and T helper cell activity, including the secretionof cytokines. In an embodiment, the intracellular signaling domain cancomprise a primary intracellular signaling domain. Exemplary primaryintracellular signaling domains include those derived from the moleculesresponsible for primary stimulation, or antigen dependent simulation. Inan embodiment, the intracellular signaling domain can comprise acostimulatory intracellular domain. Exemplary costimulatoryintracellular signaling domains include those derived from moleculesresponsible for costimulatory signals, or antigen independentstimulation.

A primary intracellular signaling domain can comprise an ITAM(“immunoreceptor tyrosine-based activation motif”). Examples of ITAMcontaining primary cytoplasmic signaling sequences include, but are notlimited to, those derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma,CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d DAP10 andDAP12.

The term “costimulatory molecule” refers to the cognate binding partneron a T-cell that specifically binds with a costimulatory ligand, therebymediating a costimulatory response by the T-cell, such as, but notlimited to, proliferation. Costimulatory molecules are cell surfacemolecules other than antigen receptors or their ligands that arerequired for an efficient immune response. Costimulatory moleculesinclude, but are not limited to an MHC class 1 molecule, BTLA and a Tollligand receptor, as well as DAP10, DAP12, CD30, LIGHT, OX40, CD2, CD27,CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18) and 4-1BB (CD137). A costimulatoryintracellular signaling domain can be the intracellular portion of acostimulatory molecule. A costimulatory molecule can be represented inthe following protein families: TNF receptor proteins,Immunoglobulin-like proteins, cytokine receptors, integrins, signalinglymphocytic activation molecules (SLAM proteins), and activating NK cellreceptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137),OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7,NKp80, CD160, B7-H3, and a ligand that specifically binds with CD83, andthe like. The intracellular signaling domain can comprise the entireintracellular portion, or the entire native intracellular signalingdomain, of the molecule from which it is derived, or a functionalfragment thereof. The term “4-1BB” refers to a member of the TNFRsuperfamily with an amino acid sequence provided as GenBank Acc. No.AAA62478.2, or the equivalent residues from a non-human species, e.g.,mouse, rodent, monkey, ape and the like; and a “4-1BB costimulatorydomain” is defined as amino acid residues 214-255 of GenBank Acc. No.AAA62478.2, or equivalent residues from non-human species, e.g., mouse,rodent, monkey, ape and the like.

The term “encoding” refers to the inherent property of specificsequences of nucleotides in a polynucleotide, such as a gene, a cDNA, oran mRNA, to serve as templates for synthesis of other polymers andmacromolecules in biological processes having either a defined sequenceof nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence ofamino acids and the biological properties resulting therefrom. Thus, agene, cDNA, or RNA, encodes a protein if transcription and translationof mRNA corresponding to that gene produces the protein in a cell orother biological system. Both the coding strand, the nucleotide sequenceof which is identical to the mRNA sequence and is usually provided insequence listings, and the non-coding strand, used as the template fortranscription of a gene or cDNA, can be referred to as encoding theprotein or other product of that gene or cDNA.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or an RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain one or more introns.

The term “effective amount” or “therapeutically effective amount” areused interchangeably herein, and refer to an amount of a compound,formulation, material, or composition, as described herein effective toachieve a particular biological or therapeutic result.

The term “endogenous” refers to any material from or produced inside anorganism, cell, tissue or system.

The term “exogenous” refers to any material introduced from or producedoutside an organism, cell, tissue or system.

The term “expression” refers to the transcription and/or translation ofa particular nucleotide sequence driven by a promoter.

The term “transfer vector” refers to a composition of matter whichcomprises an isolated nucleic acid and which can be used to deliver theisolated nucleic acid to the interior of a cell. Numerous vectors areknown in the art including, but not limited to, linear polynucleotides,polynucleotides associated with ionic or amphiphilic compounds,plasmids, and viruses. Thus, the term “transfer vector” includes anautonomously replicating plasmid or a virus. The term should also beconstrued to further include non-plasmid and non-viral compounds whichfacilitate transfer of nucleic acid into cells, such as, for example, apolylysine compound, liposome, and the like. Examples of viral transfervectors include, but are not limited to, adenoviral vectors,adeno-associated virus vectors, retroviral vectors, lentiviral vectors,and the like.

The term “expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, including cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., lentiviruses, retroviruses, adenoviruses, andadeno-associated viruses) that incorporate the recombinantpolynucleotide.

The term “lentivirus” refers to a genus of the Retroviridae family.Lentiviruses are unique among the retroviruses in being able to infectnon-dividing cells; they can deliver a significant amount of geneticinformation into the DNA of the host cell, so they are one of the mostefficient methods of a gene delivery vector. HIV, SIV, and FIV are allexamples of lentiviruses.

The term “lentiviral vector” refers to a vector derived from at least aportion of a lentivirus genome, including especially a self-inactivatinglentiviral vector as provided in Milone et al., Mol. Ther. 17(8):1453-1464 (2009). Other examples of lentivirus vectors that may be usedin the clinic, include but are not limited to, e.g., the LENTIVECTORmgene delivery technology from Oxford BioMedica, the LENTIMAX™ vectorsystem from Lentigen, and the like. Nonclinical types of lentiviralvectors are also available and would be known to one skilled in the art.

The term “homologous” or “identity” refers to the subunit sequenceidentity between two polymeric molecules, e.g., between two nucleic acidmolecules, such as, two DNA molecules or two RNA molecules, or betweentwo polypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same monomeric subunit; e.g., if a positionin each of two DNA molecules is occupied by adenine, then they arehomologous or identical at that position. The homology between twosequences is a direct function of the number of matching or homologouspositions; e.g., if half (e.g., five positions in a polymer ten subunitsin length) of the positions in two sequences are homologous, the twosequences are 50% homologous; if 90% of the positions (e.g., 9 of 10),are matched or homologous, the two sequences are 90% homologous.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.

For the most part, humanized antibodies and antibody fragments thereofare human immunoglobulins (recipient antibody or antibody fragment) inwhich residues from a complementary-determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity, and capacity. In some instances, Fv frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, a humanizedantibody/antibody fragment can comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. These modifications can further refine and optimize antibodyor antibody fragment performance. In general, the humanized antibody orantibody fragment thereof will comprise substantially all of at leastone, and typically two, variable domains, in which all or substantiallyall of the CDR regions correspond to those of a non-human immunoglobulinand all or a significant portion of the FR regions are those of a humanimmunoglobulin sequence. The humanized antibody or antibody fragment canalso comprise at least a portion of an immunoglobulin constant region(Fc), typically that of a human immunoglobulin. For further details, seeJones et al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332:323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

“Human” or “fully human” refers to an immunoglobulin, such as anantibody or antibody fragment, where the whole molecule is of humanorigin or consists of an amino acid sequence identical to a human formof the antibody or immunoglobulin.

The term “isolated” means altered or removed from the natural state. Forexample, a nucleic acid or a peptide naturally present in a livinganimal is not “isolated,” but the same nucleic acid or peptide partiallyor completely separated from the coexisting materials of its naturalstate is “isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

The term “operably linked” or “transcriptional control” refers tofunctional linkage between a regulatory sequence and a heterologousnucleic acid sequence resulting in expression of the latter. Forexample, a first nucleic acid sequence is operably linked with a secondnucleic acid sequence when the first nucleic acid sequence is placed ina functional relationship with the second nucleic acid sequence. Forinstance, a promoter is operably linked to a coding sequence if thepromoter affects the transcription or expression of the coding sequence.Operably linked DNA sequences can be contiguous with each other and,e.g., where necessary to join two protein coding regions, are in thesame reading frame.

The term “parenteral” administration of an immunogenic compositionincludes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular(i.m.), or intrasternal injection, intratumoral, or infusion techniques.

The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleicacids (DNA) or ribonucleic acids (RNA) and polymers thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogues of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions), alleles,orthologs, SNPs, and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991);Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini etal., Mol. Cell. Probes 8:91-98 (1994)).

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. A polypeptide includes a natural peptide, arecombinant peptide, or a combination thereof.

The term “promoter” refers to a DNA sequence recognized by thetranscription machinery of the cell, or introduced synthetic machinery,required to initiate the specific transcription of a polynucleotidesequence.

The term “promoter/regulatory sequence” refers to a nucleic acidsequence which is required for expression of a gene product operablylinked to the promoter/regulatory sequence. In some instances, thissequence may be the core promoter sequence and in other instances, thissequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product.

The promoter/regulatory sequence may, for example, be one whichexpresses the gene product in a tissue specific manner.

The term “constitutive” promoter refers to a nucleotide sequence which,when operably linked with a polynucleotide which encodes or specifies agene product, causes the gene product to be produced in a cell undermost or all physiological conditions of the cell.

The term “inducible” promoter refers to a nucleotide sequence which,when operably linked with a polynucleotide which encodes or specifies agene product, causes the gene product to be produced in a cellsubstantially only when an inducer which corresponds to the promoter ispresent in the cell.

The term “tissue-specific” promoter refers to a nucleotide sequencewhich, when operably linked with a polynucleotide encodes or specifiedby a gene, causes the gene product to be produced in a cellsubstantially only if the cell is a cell of the tissue typecorresponding to the promoter.

The terms “linker” and “flexible polypeptide linker” as used in thecontext of a scFv refers to a peptide linker that consists of aminoacids such as glycine and/or serine residues used alone or incombination, to link variable heavy and variable light chain regionstogether. In one embodiment, the flexible polypeptide linker is aGly/Ser linker and comprises the amino acid sequence(Gly-Gly-Gly-Ser)_(n), where n is a positive integer equal to or greaterthan 1. For example, n=1, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9 andn=10. In one embodiment, the flexible polypeptide linkers include, butare not limited to, (Gly₄Ser)₄ or (Gly₄Ser)₃. In another embodiment, thelinkers include multiple repeats of (Gy₂Ser), (GlySer) or (Gly₃Ser).Also included within the scope of the invention are linkers described inWO2012/138475 (incorporated herein by reference). In some instances, thelinker sequence comprises a long linker (LL) sequence. In someinstances, the long linker sequence comprises (G₄S)_(n), wherein n=2 to4. In some instances, the linker sequence comprises a short linker (SL)sequence. In some instances, the short linker sequence comprises(G₄S)_(n), wherein n=1 to 3.

As used herein, a 5′ cap (also termed an RNA cap, an RNA7-methylguanosine cap or an RNA m7G cap) is a modified guaninenucleotide that has been added to the “front” or 5′ end of a eukaryoticmessenger RNA shortly after the start of transcription. The 5′ capconsists of a terminal group which is linked to the first transcribednucleotide. Its presence is critical for recognition by the ribosome andprotection from RNases. Cap addition is coupled to transcription, andoccurs co-transcriptionally, such that each influences the other.Shortly after the start of transcription, the 5′ end of the mRNA beingsynthesized is bound by a cap-synthesizing complex associated with RNApolymerase. This enzymatic complex catalyzes the chemical reactions thatare required for mRNA capping. Synthesis proceeds as a multi-stepbiochemical reaction. The capping moiety can be modified to modulatefunctionality of mRNA such as its stability or efficiency oftranslation.

As used herein, “in vitro transcribed RNA” refers to RNA, preferablymRNA, which has been synthesized in vitro. Generally, the in vitrotranscribed RNA is generated from an in vitro transcription vector. Thein vitro transcription vector comprises a template that is used togenerate the in vitro transcribed RNA.

As used herein, a “poly(A)” is a series of adenosines attached bypolyadenylation to the mRNA. In the preferred embodiment of a constructfor transient expression, the polyA is between 50 and 5000, preferablygreater than 64, more preferably greater than 100, most preferablygreater than 300 or 400. Poly(A) sequences can be modified chemically orenzymatically to modulate mRNA functionality such as localization,stability or efficiency of translation.

As used herein, “polyadenylation” refers to the covalent linkage of apolyadenylyl moiety, or its modified variant, to a messenger RNAmolecule. In eukaryotic organisms, most messenger RNA (mRNA) moleculesare polyadenylated at the 3′ end. The 3′ poly(A) tail is a long sequenceof adenine nucleotides (often several hundred) added to the pre-mRNAthrough the action of an enzyme, polyadenylate polymerase. In highereukaryotes, the poly(A) tail is added onto transcripts that contain aspecific sequence, the polyadenylation signal. The poly(A) tail and theprotein bound to it aid in protecting mRNA from degradation byexonucleases. Polyadenylation is also important for transcriptiontermination, export of the mRNA from the nucleus, and translation.Polyadenylation occurs in the nucleus immediately after transcription ofDNA into RNA, but additionally can also occur later in the cytoplasm.After transcription has been terminated, the mRNA chain is cleavedthrough the action of an endonuclease complex associated with RNApolymerase. The cleavage site is usually characterized by the presenceof the base sequence AAUAAA near the cleavage site. After the mRNA hasbeen cleaved, adenosine residues are added to the free 3′ end at thecleavage site.

As used herein, “transient” refers to expression of a non-integratedtransgene for a period of hours, days or weeks, wherein the period oftime of expression is less than the period of time for expression of thegene if integrated into the genome or contained within a stable plasmidreplicon in the host cell.

The term “signal transduction pathway” refers to the biochemicalrelationship between a variety of signal transduction molecules thatplay a role in the transmission of a signal from one portion of a cellto another portion of a cell. The phrase “cell surface receptor”includes molecules and complexes of molecules capable of receiving asignal and transmitting signal across the membrane of a cell.

The term “subject” is intended to include living organisms in which animmune response can be elicited (e.g., mammals, human).

The term, a “substantially purified” cell refers to a cell that isessentially free of other cell types. A substantially purified cell alsorefers to a cell which has been separated from other cell types withwhich it is normally associated in its naturally occurring state. Insome instances, a population of substantially purified cells refers to ahomogenous population of cells. In other instances, this term referssimply to cell that have been separated from the cells with which theyare naturally associated in their natural state. In some aspects, thecells are cultured in vitro. In other aspects, the cells are notcultured in vitro.

The term “therapeutic” as used herein means a treatment. A therapeuticeffect is obtained by reduction, suppression, remission, or eradicationof a disease state.

The term “prophylaxis” as used herein means the prevention of orprotective treatment for a disease or disease state.

In the context of the present invention, “tumor antigen” or“hyperproliferative disorder antigen” or “antigen associated with ahyperproliferative disorder” refers to antigens that are common tospecific hyperproliferative disorders. In certain aspects, thehyperproliferative disorder antigens of the present invention arederived from, cancers including but not limited to primary or metastaticmelanoma

mesothelioma, renal cell carcinoma, stomach cancer, breast cancer, lungcancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer,brain cancer, liver cancer, pancreatic cancer, kidney, endometrial, andstomach cancer.

In some instances, the disease is a cancer selected from the groupconsisting of mesothelioma, papillary serous ovarian adenocarcinoma,clear cell ovarian carcinoma, mixed Mullerian ovarian carcinoma,endometroid mucinous ovarian carcinoma, malignant pleural disease,pancreatic adenocarcinoma, ductal pancreatic adenocarcinoma, uterineserous carcinoma, lung adenocarcinoma, extrahepatic bile duct carcinoma,gastric adenocarcinoma, esophageal adenocarcinoma, colorectaladenocarcinoma, breast adenocarcinoma, a disease associated withmesothelin expression, and combinations thereof, a disease associatedwith mesothelin expression, and combinations thereof.

The term “transfected” or “transformed” or “transduced” refers to aprocess by which exogenous nucleic acid is transferred or introducedinto the host cell. A “transfected” or “transformed” or “transduced”cell is one which has been transfected, transformed or transduced withexogenous nucleic acid. The cell includes the primary subject cell andits progeny.

The term “specifically binds,” refers to an antibody, an antibodyfragment or a specific ligand, which recognizes and binds a cognatebinding partner (e.g., mesothelin) present in a sample, but which doesnot necessarily and substantially recognize or bind other molecules inthe sample.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Asanother example, a range such as 95-99% identity, includes somethingwith 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This appliesregardless of the breadth of the range.

DESCRIPTION

Provided herein are compositions of matter and methods of use for thetreatment of a disease such as cancer, using T-cell receptor (TCR)fusion proteins. As used herein, a “T-cell receptor (TCR) fusionprotein” or “TFP” includes a recombinant polypeptide derived from thevarious polypeptides comprising the TCR that is generally capable of i)binding to a surface antigen on target cells and ii) interacting withother polypeptide components of the intact TCR complex, typically whenco-located in or on the surface of a T-cell. As provided herein, TFPsprovide substantial benefits as compared to Chimeric Antigen Receptors.The term “Chimeric Antigen Receptor” or alternatively a “CAR” refers toa recombinant polypeptide comprising an extracellular antigen bindingdomain in the form of a scFv, a transmembrane domain, and cytoplasmicsignaling domains (also referred to herein as “an intracellularsignaling domains”) comprising a functional signaling domain derivedfrom a stimulatory molecule as defined below. Generally, the centralintracellular signaling domain of a CAR is derived from the CD3 zetachain that is normally found associated with the TCR complex. The CD3zeta signaling domain can be fused with one or more functional signalingdomains derived from at least one co-stimulatory molecule such as 4-1BB(i.e., CD137), CD27 and/or CD28.

T-Cell Receptor (TCR) Fusion Proteins (TFP)

The present invention encompasses recombinant DNA constructs encodingTFPs, wherein the TFP comprises an antibody fragment that bindsspecifically to mesothelin, e.g., human mesothelin, wherein the sequenceof the antibody fragment is contiguous with and in the same readingframe as a nucleic acid sequence encoding a TCR subunit or portionthereof. The TFPs provided herein are able to associate with one or moreendogenous (or alternatively, one or more exogenous, or a combination ofendogenous and exogenous) TCR subunits in order to form a functional TCRcomplex.

In one aspect, the TFP of the invention comprises a target-specificbinding element otherwise referred to as an antigen binding domain. Thechoice of moiety depends upon the type and number of target antigen thatdefine the surface of a target cell. For example, the antigen bindingdomain may be chosen to recognize a target antigen that acts as a cellsurface marker on target cells associated with a particular diseasestate. Thus, examples of cell surface markers that may act as targetantigens for the antigen binding domain in a TFP of the inventioninclude those associated with viral, bacterial and parasitic infections;autoimmune diseases; and cancerous diseases (e.g., malignant diseases).

In one aspect, the TFP-mediated T-cell response can be directed to anantigen of interest by way of engineering an antigen-binding domain intothe TFP that specifically binds a desired antigen.

In one aspect, the portion of the TFP comprising the antigen bindingdomain comprises an antigen binding domain that targets mesothelin. Inone aspect, the antigen binding domain targets human mesothelin.

The antigen binding domain can be any domain that binds to the antigenincluding but not limited to a monoclonal antibody, a polyclonalantibody, a recombinant antibody, a human antibody, a humanizedantibody, and a functional fragment thereof, including but not limitedto a single-domain antibody such as a heavy chain variable domain(V_(H)), a light chain variable domain (V_(L)) and a variable domain(V_(HH)) of a camelid derived nanobody, and to an alternative scaffoldknown in the art to function as antigen binding domain, such as arecombinant fibronectin domain, anticalin, DARPIN and the like. Likewisea natural or synthetic ligand specifically recognizing and binding thetarget antigen can be used as antigen binding domain for the TFP. Insome instances, it is beneficial for the antigen binding domain to bederived from the same species in which the TFP will ultimately be usedin. For example, for use in humans, it may be beneficial for the antigenbinding domain of the TFP to comprise human or humanized residues forthe antigen binding domain of an antibody or antibody fragment.

Thus, in one aspect, the antigen-binding domain comprises a humanized orhuman antibody or an antibody fragment, or a murine antibody or antibodyfragment. In one embodiment, the humanized or human anti-mesothelinbinding domain comprises one or more (e.g., all three) light chaincomplementary determining region 1 (LC CDR1), light chain complementarydetermining region 2 (LC CDR2), and light chain complementarydetermining region 3 (LC CDR3) of a humanized or human anti-mesothelinbinding domain described herein, and/or one or more (e.g., all three)heavy chain complementary determining region 1 (HC CDR1), heavy chaincomplementary determining region 2 (HC CDR2), and heavy chaincomplementary determining region 3 (HC CDR3) of a humanized or humananti-mesothelin binding domain described herein, e.g., a humanized orhuman anti-mesothelin binding domain comprising one or more, e.g., allthree, LC CDRs and one or more, e.g., all three, HC CDRs. In oneembodiment, the humanized or human anti-mesothelin binding domaincomprises one or more (e.g., all three) heavy chain complementarydetermining region 1 (HC CDR1), heavy chain complementary determiningregion 2 (HC CDR2), and heavy chain complementary determining region 3(HC CDR3) of a humanized or human anti-mesothelin binding domaindescribed herein, e.g., the humanized or human anti-mesothelin bindingdomain has two variable heavy chain regions, each comprising a HC CDR1,a HC CDR2 and a HC CDR3 described herein. In one embodiment, thehumanized or human anti-mesothelin binding domain comprises a humanizedor human light chain variable region described herein and/or a humanizedor human heavy chain variable region described herein. In oneembodiment, the humanized or human anti-mesothelin binding domaincomprises a humanized heavy chain variable region described herein,e.g., at least two humanized or human heavy chain variable regionsdescribed herein. In one embodiment, the anti-mesothelin binding domainis a scFv comprising a light chain and a heavy chain of an amino acidsequence provided herein. In an embodiment, the anti-mesothelin bindingdomain (e.g., a scFv) comprises: a light chain variable regioncomprising an amino acid sequence having at least one, two or threemodifications (e.g., substitutions) but not more than 30, 20 or 10modifications (e.g., substitutions) of an amino acid sequence of a lightchain variable region provided herein, or a sequence with 95-99%identity with an amino acid sequence provided herein; and/or a heavychain variable region comprising an amino acid sequence having at leastone, two or three modifications (e.g., substitutions) but not more than30, 20 or 10 modifications (e.g., substitutions) of an amino acidsequence of a heavy chain variable region provided herein, or a sequencewith 95-99% identity to an amino acid sequence provided herein. In oneembodiment, the humanized or human anti-mesothelin binding domain is ascFv, and a light chain variable region comprising an amino acidsequence described herein, is attached to a heavy chain variable regioncomprising an amino acid sequence described herein, via a linker, e.g.,a linker described herein. In one embodiment, the humanizedanti-mesothelin binding domain includes a (Gly₄-Ser)_(n) linker, whereinn is 1, 2, 3, 4, 5, or 6, preferably 3 or 4. The light chain variableregion and heavy chain variable region of a scFv can be, e.g., in any ofthe following orientations: light chain variable region-linker-heavychain variable region or heavy chain variable region-linker-light chainvariable region. In some instances, the linker sequence comprises a longlinker (LL) sequence. In some instances, the long linker sequencecomprises (G₄S)_(n), wherein n=2 to 4. In some instances, the linkersequence comprises a short linker (SL) sequence. In some instances, theshort linker sequence comprises (G₄S)_(n), wherein n=1 to 3.

In some aspects, a non-human antibody is humanized, where specificsequences or regions of the antibody are modified to increase similarityto an antibody naturally produced in a human or fragment thereof. In oneaspect, the antigen binding domain is humanized.

A humanized antibody can be produced using a variety of techniques knownin the art, including but not limited to, CDR-grafting (see, e.g.,European Patent No. EP 239,400; International Publication No. WO91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089, eachof which is incorporated herein in its entirety by reference), veneeringor resurfacing (see, e.g., European Patent Nos. EP 592,106 and EP519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnickaet al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al.,1994, PNAS, 91:969-973, each of which is incorporated herein by itsentirety by reference), chain shuffling (see, e.g., U.S. Pat. No.5,565,332, which is incorporated herein in its entirety by reference),and techniques disclosed in, e.g., U.S. Patent Application PublicationNo. US2005/0042664, U.S. Patent Application Publication No.US2005/0048617, U.S. Pat. Nos. 6,407,213, 5,766,886, InternationalPublication No. WO 93/017105, Tan et al., J. Immunol., 169:1119-25(2002), Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al.,Methods, 20(3):267-79 (2000), Baca et al., J. Biol. Chem.,272(16):10678-84 (1997), Roguska et al., Protein Eng., 9(10):895-904(1996), Couto et al., Cancer Res., 55 (23 Supp):5973s-5977s (1995),Couto et al., Cancer Res., 55(8):1717-22 (1995), Sandhu J S, Gene,150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol., 235(3):959-73(1994), each of which is incorporated herein in its entirety byreference. Often, framework residues in the framework regions will besubstituted with the corresponding residue from the CDR donor antibodyto alter, for example improve, antigen binding. These frameworksubstitutions are identified by methods well-known in the art, e.g., bymodeling of the interactions of the CDR and framework residues toidentify framework residues important for antigen binding and sequencecomparison to identify unusual framework residues at particularpositions (see, e.g., Queen et al., U.S. Pat. No. 5,585,089; andRiechmann et al., 1988, Nature, 332:323, which are incorporated hereinby reference in their entireties.)

A humanized antibody or antibody fragment has one or more amino acidresidues remaining in it from a source which is nonhuman. These nonhumanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. As providedherein, humanized antibodies or antibody fragments comprise one or moreCDRs from nonhuman immunoglobulin molecules and framework regionswherein the amino acid residues comprising the framework are derivedcompletely or mostly from human germline. Multiple techniques forhumanization of antibodies or antibody fragments are well-known in theart and can essentially be performed following the method of Winter andco-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al.,Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536(1988)), by substituting rodent CDRs or CDR sequences for thecorresponding sequences of a human antibody, i.e., CDR-grafting (EP239,400; PCT Publication No. WO 91/09967; and U.S. Pat. Nos. 4,816,567;6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640, the contents ofwhich are incorporated herein by reference in their entirety). In suchhumanized antibodies and antibody fragments, substantially less than anintact human variable domain has been substituted by the correspondingsequence from a nonhuman species. Humanized antibodies are often humanantibodies in which some CDR residues and possibly some framework (FR)residues are substituted by residues from analogous sites in rodentantibodies. Humanization of antibodies and antibody fragments can alsobe achieved by veneering or resurfacing (EP 592,106; EP 519,596; Padlan,1991, Molecular Immunology, 28(4/5):489-498; Studnicka et al., ProteinEngineering, 7(6):805-814 (1994); and Roguska et al., PNAS, 91:969-973(1994)) or chain shuffling (U.S. Pat. No. 5,565,332), the contents ofwhich are incorporated herein by reference in their entirety.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is to reduce antigenicity. Accordingto the so-called “best-fit” method, the sequence of the variable domainof a rodent antibody is screened against the entire library of knownhuman variable-domain sequences. The human sequence which is closest tothat of the rodent is then accepted as the human framework (FR) for thehumanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothiaet al., J. Mol. Biol., 196:901 (1987), the contents of which areincorporated herein by reference herein in their entirety). Anothermethod uses a particular framework derived from the consensus sequenceof all human antibodies of a particular subgroup of light or heavychains. The same framework may be used for several different humanizedantibodies (see, e.g., Nicholson et al. Mol. Immun. 34 (16-17):1157-1165 (1997); Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285(1992); Presta et al., J. Immunol., 151:2623 (1993), the contents ofwhich are incorporated herein by reference herein in their entirety). Insome embodiments, the framework region, e.g., all four frameworkregions, of the heavy chain variable region are derived from aV_(H)4-4-59 germline sequence. In one embodiment, the framework regioncan comprise, one, two, three, four or five modifications, e.g.,substitutions, e.g., from the amino acid at the corresponding murinesequence. In one embodiment, the framework region, e.g., all fourframework regions of the light chain variable region are derived from aVK3-1.25 germline sequence. In one embodiment, the framework region cancomprise, one, two, three, four or five modifications, e.g.,substitutions, e.g., from the amino acid at the corresponding murinesequence.

In some aspects, the portion of a TFP composition of the invention thatcomprises an antibody fragment is humanized with retention of highaffinity for the target antigen and other favorable biologicalproperties. According to one aspect of the invention, humanizedantibodies and antibody fragments are prepared by a process of analysisof the parental sequences and various conceptual humanized productsusing three-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, e.g., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind the target antigen. In this way, FR residues canbe selected and combined from the recipient and import sequences so thatthe desired antibody or antibody fragment characteristic, such asincreased affinity for the target antigen, is achieved. In general, theCDR residues are directly and most substantially involved in influencingantigen binding.

A humanized antibody or antibody fragment may retain a similar antigenicspecificity as the original antibody, e.g., in the present invention,the ability to bind human mesothelin. In some embodiments, a humanizedantibody or antibody fragment may have improved affinity and/orspecificity of binding to human mesothelin.

In one aspect, the anti-mesothelin binding domain is characterized byparticular functional features or properties of an antibody or antibodyfragment. For example, in one aspect, the portion of a TFP compositionof the invention that comprises an antigen binding domain specificallybinds human mesothelin. In one aspect, the antigen binding domain hasthe same or a similar binding specificity to human mesothelin as theFMC63 scFv described in Nicholson et al. Mol. Immun. 34 (16-17):1157-1165 (1997). In one aspect, the invention relates to an antigenbinding domain comprising an antibody or antibody fragment, wherein theantibody binding domain specifically binds to a mesothelin protein orfragment thereof, wherein the antibody or antibody fragment comprises avariable light chain and/or a variable heavy chain that includes anamino acid sequence provided herein. In certain aspects, the scFv iscontiguous with and in the same reading frame as a leader sequence.

In one aspect, the anti-mesothelin binding domain is a fragment, e.g., asingle chain variable fragment (scFv). In one aspect, theanti-mesothelin binding domain is a Fv, a Fab, a (Fab′)₂, or abi-functional (e.g. bi-specific) hybrid antibody (e.g., Lanzavecchia etal., Eur. J. Immunol. 17, 105 (1987)).

In one aspect, the antibodies and fragments thereof disclosed hereinbind a mesothelin protein with wild-type or enhanced affinity.

Also provided herein are methods for obtaining an antibody antigenbinding domain specific for a target antigen (e.g., mesothelin or anytarget antigen described elsewhere herein for targets of fusion moietybinding domains), the method comprising providing by way of addition,deletion, substitution or insertion of one or more amino acids in theamino acid sequence of a V_(H) domain set out herein a V_(H) domainwhich is an amino acid sequence variant of the V_(H) domain, optionallycombining the V_(H) domain thus provided with one or more V_(L) domains,and testing the V_(H) domain or V_(H)V_(L) combination or combinationsto identify a specific binding member or an antibody antigen bindingdomain specific for a target antigen of interest (e.g., mesothelin) andoptionally with one or more desired properties.

In some instances, V_(H) domains and scFvs can be prepared according tomethod known in the art (see, for example, Bird et al., (1988) Science242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA85:5879-5883). scFv molecules can be produced by linking V_(H) and V_(L)regions together using flexible polypeptide linkers. The scFv moleculescomprise a linker (e.g., a Ser-Gly linker) with an optimized lengthand/or amino acid composition. The linker length can greatly affect howthe variable regions of a scFv fold and interact. In fact, if a shortpolypeptide linker is employed (e.g., between 5-10 amino acids)intra-chain folding is prevented. Inter-chain folding is also requiredto bring the two variable regions together to form a functional epitopebinding site. In some instances, the linker sequence comprises a longlinker (LL) sequence. In some instances, the long linker sequencecomprises (G₄S)_(n), wherein n=2 to 4. In some instances, the linkersequence comprises a short linker (SL) sequence. In some instances, theshort linker sequence comprises (G₄S)_(n), wherein n=1 to 3. Forexamples of linker orientation and size see, e.g., Hollinger et al. 1993Proc Natl Acad. Sci. U.S.A. 90:6444-6448, U.S. Pat. No. 7,695,936, U.S.Patent Application Publication Nos. 20050100543 and 20050175606, and PCTPublication Nos. WO2006/020258 and WO2007/024715, all of which areincorporated herein by reference.

A scFv can comprise a linker of about 10, 11, 12, 13, 14, 15 or greaterthan 15 residues between its V_(L) and V_(H) regions. The linkersequence may comprise any naturally occurring amino acid. In someembodiments, the linker sequence comprises amino acids glycine andserine. In another embodiment, the linker sequence comprises sets ofglycine and serine repeats such as (Gy₄Ser)_(n), where n is a positiveinteger equal to or greater than 1. In one embodiment, the linker can be(Gy₄Ser)₄ or (Gy₄Ser)₃. Variation in the linker length may retain orenhance activity, giving rise to superior efficacy in activity studies.In some instances, the linker sequence comprises a long linker (LL)sequence. In some instances, the long linker sequence comprises(G₄S)_(n), wherein n=2 to 4. In some instances, the linker sequencecomprises a short linker (SL) sequence. In some instances, the shortlinker sequence comprises (G₄S)_(n), wherein n=1 to 3.

Stability and Mutations

The stability of an anti-mesothelin binding domain, e.g., scFv molecules(e.g., soluble scFv) can be evaluated in reference to the biophysicalproperties (e.g., thermal stability) of a conventional control scFvmolecule or a full length antibody. In one embodiment, the humanized orhuman scFv has a thermal stability that is greater than about 0.1, about0.25, about 0.5, about 0.75, about 1, about 1.25, about 1.5, about 1.75,about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5,about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5,about 9, about 9.5, about 10 degrees, about 11 degrees, about 12degrees, about 13 degrees, about 14 degrees, or about 15 degrees Celsiusthan a parent scFv in the described assays.

The improved thermal stability of the anti-mesothelin binding domain,e.g., scFv is subsequently conferred to the entire mesothelin-TFPconstruct, leading to improved therapeutic properties of theanti-mesothelin TFP construct. The thermal stability of theanti-mesothelin binding domain, e.g., scFv can be improved by at leastabout 2° C. or 3° C. as compared to a conventional antibody. In oneembodiment, the anti-mesothelin binding domain, e.g., scFv has a 1° C.improved thermal stability as compared to a conventional antibody. Inanother embodiment, the anti-mesothelin binding domain, e.g., scFv has a2° C. improved thermal stability as compared to a conventional antibody.In another embodiment, the scFv has a 4° C., 5° C., 6° C., 7° C., 8° C.,9° C., 10° C., 11° C., 12° C., 13° C., 14° C., or 15° C. improvedthermal stability as compared to a conventional antibody. Comparisonscan be made, for example, between the scFv molecules disclosed hereinand scFv molecules or Fab fragments of an antibody from which the scFvV_(H) and V_(L) were derived. Thermal stability can be measured usingmethods known in the art. For example, in one embodiment, T_(M) can bemeasured. Methods for measuring T_(M) and other methods of determiningprotein stability are described below.

Mutations in scFv (arising through humanization or mutagenesis of thesoluble scFv) alter the stability of the scFv and improve the overallstability of the scFv and the anti-mesothelin TFP construct. Stabilityof the humanized scFv is compared against the murine scFv usingmeasurements such as T_(M), temperature denaturation and temperatureaggregation. In one embodiment, the anti-mesothelin binding domain,e.g., a scFv, comprises at least one mutation arising from thehumanization process such that the mutated scFv confers improvedstability to the anti-mesothelin TFP construct. In another embodiment,the anti-mesothelin binding domain, e.g., scFv comprises at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10 mutations arising from the humanization processsuch that the mutated scFv confers improved stability to themesothelin-TFP construct.

In one aspect, the antigen binding domain of the TFP comprises an aminoacid sequence that is homologous to an antigen binding domain amino acidsequence described herein, and the antigen binding domain retains thedesired functional properties of the anti-mesothelin antibody fragmentsdescribed herein. In one specific aspect, the TFP composition of theinvention comprises an antibody fragment. In a further aspect, thatantibody fragment comprises a scFv.

In various aspects, the antigen binding domain of the TFP is engineeredby modifying one or more amino acids within one or both variable regions(e.g., V_(H) and/or V_(L)), for example within one or more CDR regionsand/or within one or more framework regions. In one specific aspect, theTFP composition of the invention comprises an antibody fragment. In afurther aspect, that antibody fragment comprises a scFv.

It will be understood by one of ordinary skill in the art that theantibody or antibody fragment of the invention may further be modifiedsuch that they vary in amino acid sequence (e.g., from wild-type), butnot in desired activity. For example, additional nucleotidesubstitutions leading to amino acid substitutions at “non-essential”amino acid residues may be made to the protein. For example, anonessential amino acid residue in a molecule may be replaced withanother amino acid residue from the same side chain family. In anotherembodiment, a string of amino acids can be replaced with a structurallysimilar string that differs in order and/or composition of side chainfamily members, e.g., a conservative substitution, in which an aminoacid residue is replaced with an amino acid residue having a similarside chain, may be made.

Families of amino acid residues having similar side chains have beendefined in the art, including basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine).

Percent identity in the context of two or more nucleic acids orpolypeptide sequences refers to two or more sequences that are the same.Two sequences are “substantially identical” if two sequences have aspecified percentage of amino acid residues or nucleotides that are thesame (e.g., 60% identity, optionally 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity over a specifiedregion, or, when not specified, over the entire sequence), when comparedand aligned for maximum correspondence over a comparison window, ordesignated region as measured using one of the following sequencecomparison algorithms or by manual alignment and visual inspection.Optionally, the identity exists over a region that is at least about 50nucleotides (or 10 amino acids) in length, or more preferably over aregion that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 ormore amino acids) in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters. Methods of alignment of sequences forcomparison are well known in the art. Optimal alignment of sequences forcomparison can be conducted, e.g., by the local homology algorithm ofSmith and Waterman, (1970) Adv. Appl. Math. 2:482c, by the homologyalignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol.48:443, by the search for similarity method of Pearson and Lipman,(1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by manual alignment and visualinspection (see, e.g., Brent et al., (2003) Current Protocols inMolecular Biology). Two examples of algorithms that are suitable fordetermining percent sequence identity and sequence similarity are theBLAST and BLAST 2.0 algorithms, which are described in Altschul et al.,(1977) Nuc. Acids Res. 25:3389-3402; and Altschul et al., (1990) J. Mol.Biol. 215:403-410, respectively. Software for performing BLAST analysesis publicly available through the National Center for BiotechnologyInformation.

In one aspect, the present invention contemplates modifications of thestarting antibody or fragment (e.g., scFv) amino acid sequence thatgenerate functionally equivalent molecules. For example, the V_(H) orV_(L) of an anti-mesothelin binding domain, e.g., scFv, comprised in theTFP can be modified to retain at least about 70%, 71%. 72%. 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity of thestarting V_(H) or V_(L) framework region of the anti-mesothelin bindingdomain, e.g., scFv. The present invention contemplates modifications ofthe entire TFP construct, e.g., modifications in one or more amino acidsequences of the various domains of the TFP construct in order togenerate functionally equivalent molecules. The TFP construct can bemodified to retain at least about 70%, 71%. 72%. 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity of the startingTFP construct.

Extracellular Domain

The extracellular domain may be derived either from a natural or from arecombinant source. Where the source is natural, the domain may bederived from any protein, but in particular a membrane-bound ortransmembrane protein. In one aspect the extracellular domain is capableof associating with the transmembrane domain. An extracellular domain ofparticular use in this invention may include at least the extracellularregion(s) of e.g., the alpha, beta or zeta chain of the T-cell receptor,or CD3 epsilon, CD3 gamma, or CD3 delta, or in alternative embodiments,CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80,CD86, CD134, CD137, CD154.

Transmembrane Domain

In general, a TFP sequence contains an extracellular domain and atransmembrane domain encoded by a single genomic sequence. Inalternative embodiments, a TFP can be designed to comprise atransmembrane domain that is heterologous to the extracellular domain ofthe TFP. A transmembrane domain can include one or more additional aminoacids adjacent to the transmembrane region, e.g., one or more amino acidassociated with the extracellular region of the protein from which thetransmembrane was derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or up to15 amino acids of the extracellular region) and/or one or moreadditional amino acids associated with the intracellular region of theprotein from which the transmembrane protein is derived (e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, 10 or up to 15 amino acids of the intracellularregion). In one aspect, the transmembrane domain is one that isassociated with one of the other domains of the TFP is used. In someinstances, the transmembrane domain can be selected or modified by aminoacid substitution to avoid binding of such domains to the transmembranedomains of the same or different surface membrane proteins, e.g., tominimize interactions with other members of the receptor complex. In oneaspect, the transmembrane domain is capable of homodimerization withanother TFP on the TFP-T-cell surface. In a different aspect the aminoacid sequence of the transmembrane domain may be modified or substitutedso as to minimize interactions with the binding domains of the nativebinding partner present in the same TFP.

The transmembrane domain may be derived either from a natural or from arecombinant source. Where the source is natural, the domain may bederived from any membrane-bound or transmembrane protein. In one aspectthe transmembrane domain is capable of signaling to the intracellulardomain(s) whenever the TFP has bound to a target. A transmembrane domainof particular use in this invention may include at least thetransmembrane region(s) of e.g., the alpha, beta or zeta chain of theT-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16,CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.

In some instances, the transmembrane domain can be attached to theextracellular region of the TFP, e.g., the antigen binding domain of theTFP, via a hinge, e.g., a hinge from a human protein. For example, inone embodiment, the hinge can be a human immunoglobulin (Ig) hinge,e.g., an IgG4 hinge, or a CD8a hinge.

Linkers

Optionally, a short oligo- or polypeptide linker, between 2 and 10 aminoacids in length may form the linkage between the transmembrane domainand the cytoplasmic region of the TFP. A glycine-serine doublet providesa particularly suitable linker. For example, in one aspect, the linkercomprises the amino acid sequence of GGGGSGGGGS (SEQ ID NO. 53). In someembodiments, the linker is encoded by a nucleotide sequence ofGGTGGCGGAGGTTCTGGAGGTGGAGGTTCC (SEQ ID NO. 54).

Cytoplasmic Domain

The cytoplasmic domain of the TFP can include an intracellular signalingdomain, if the TFP contains CD3 gamma, delta or epsilon polypeptides;TCR alpha and TCR beta subunits are generally lacking in a signalingdomain. An intracellular signaling domain is generally responsible foractivation of at least one of the normal effector functions of theimmune cell in which the TFP has been introduced. The term “effectorfunction” refers to a specialized function of a cell. Effector functionof a T-cell, for example, may be cytolytic activity or helper activityincluding the secretion of cytokines. Thus the term “intracellularsignaling domain” refers to the portion of a protein which transducesthe effector function signal and directs the cell to perform aspecialized function. While usually the entire intracellular signalingdomain can be employed, in many cases it is not necessary to use theentire chain. To the extent that a truncated portion of theintracellular signaling domain is used, such truncated portion may beused in place of the intact chain as long as it transduces the effectorfunction signal. The term intracellular signaling domain is thus meantto include any truncated portion of the intracellular signaling domainsufficient to transduce the effector function signal.

Examples of intracellular signaling domains for use in the TFP of theinvention include the cytoplasmic sequences of the T-cell receptor (TCR)and co-receptors that act in concert to initiate signal transductionfollowing antigen receptor engagement, as well as any derivative orvariant of these sequences and any recombinant sequence that has thesame functional capability.

It is known that signals generated through the TCR alone areinsufficient for full activation of naive T-cells and that a secondaryand/or costimulatory signal is required. Thus, naive T-cell activationcan be said to be mediated by two distinct classes of cytoplasmicsignaling sequences: those that initiate antigen-dependent primaryactivation through the TCR (primary intracellular signaling domains) andthose that act in an antigen-independent manner to provide a secondaryor costimulatory signal (secondary cytoplasmic domain, e.g., acostimulatory domain).

A primary signaling domain regulates primary activation of the TCRcomplex either in a stimulatory way, or in an inhibitory way. Primaryintracellular signaling domains that act in a stimulatory manner maycontain signaling motifs which are known as immunoreceptortyrosine-based activation motifs (ITAMs).

Examples of ITAMs containing primary intracellular signaling domainsthat are of particular use in the invention include those of CD3 zeta,FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22,CD79a, CD79b, and CD66d. In one embodiment, a TFP of the inventioncomprises an intracellular signaling domain, e.g., a primary signalingdomain of CD3-epsilon. In one embodiment, a primary signaling domaincomprises a modified ITAM domain, e.g., a mutated ITAM domain which hasaltered (e.g., increased or decreased) activity as compared to thenative ITAM domain. In one embodiment, a primary signaling domaincomprises a modified ITAM-containing primary intracellular signalingdomain, e.g., an optimized and/or truncated ITAM-containing primaryintracellular signaling domain. In an embodiment, a primary signalingdomain comprises one, two, three, four or more ITAM motifs.

The intracellular signaling domain of the TFP can comprise the CD3 zetasignaling domain by itself or it can be combined with any other desiredintracellular signaling domain(s) useful in the context of a TFP of theinvention. For example, the intracellular signaling domain of the TFPcan comprise a CD3 epsilon chain portion and a costimulatory signalingdomain. The costimulatory signaling domain refers to a portion of theTFP comprising the intracellular domain of a costimulatory molecule. Acostimulatory molecule is a cell surface molecule other than an antigenreceptor or its ligands that is required for an efficient response oflymphocytes to an antigen. Examples of such molecules include CD27,CD28, 4-1BB (CD137), OX40, DAP10, DAP12, CD30, CD40, PD1, ICOS,lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,NKG2C, B7-H3, and a ligand that specifically binds with CD83, and thelike. For example, CD27 costimulation has been demonstrated to enhanceexpansion, effector function, and survival of human TFP-T-cells in vitroand augments human T-cell persistence and antitumor activity in vivo(Song et al. Blood. 2012; 119(3):696-706).

The intracellular signaling sequences within the cytoplasmic portion ofthe TFP of the invention may be linked to each other in a random orspecified order. Optionally, a short oligo- or polypeptide linker, forexample, between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or10 amino acids) in length may form the linkage between intracellularsignaling sequences.

In one embodiment, a glycine-serine doublet can be used as a suitablelinker. In one embodiment, a single amino acid, e.g., an alanine, aglycine, can be used as a suitable linker.

In one aspect, the TFP-expressing cell described herein can furthercomprise a second TFP, e.g., a second TFP that includes a differentantigen binding domain, e.g., to the same target (mesothelin) or adifferent target (e.g., CD123). In one embodiment, when theTFP-expressing cell comprises two or more different TFPs, the antigenbinding domains of the different TFPs can be such that the antigenbinding domains do not interact with one another. For example, a cellexpressing a first and second TFP can have an antigen binding domain ofthe first TFP, e.g., as a fragment, e.g., a scFv, that does notassociate with the antigen binding domain of the second TFP, e.g., theantigen binding domain of the second TFP is a V_(HH).

In another aspect, the TFP-expressing cell described herein can furtherexpress another agent, e.g., an agent which enhances the activity of aTFP-expressing cell. For example, in one embodiment, the agent can be anagent which inhibits an inhibitory molecule. Inhibitory molecules, e.g.,PD1, can, in some embodiments, decrease the ability of a TFP-expressingcell to mount an immune effector response. Examples of inhibitorymolecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT,LAIR1, CD160, 2B4 and TGFR beta. In one embodiment, the agent thatinhibits an inhibitory molecule comprises a first polypeptide, e.g., aninhibitory molecule, associated with a second polypeptide that providesa positive signal to the cell, e.g., an intracellular signaling domaindescribed herein. In one embodiment, the agent comprises a firstpolypeptide, e.g., of an inhibitory molecule such as PD1, LAG3, CTLA4,CD160, BTLA, LAIR1, TIM3, 2B4 and TIGIT, or a fragment of any of these(e.g., at least a portion of an extracellular domain of any of these),and a second polypeptide which is an intracellular signaling domaindescribed herein (e.g., comprising a costimulatory domain (e.g., 4-1BB,CD27 or CD28, e.g., as described herein) and/or a primary signalingdomain (e.g., a CD3 zeta signaling domain described herein). In oneembodiment, the agent comprises a first polypeptide of PD1 or a fragmentthereof (e.g., at least a portion of an extracellular domain of PD1),and a second polypeptide of an intracellular signaling domain describedherein (e.g., a CD28 signaling domain described herein and/or a CD3 zetasignaling domain described herein). PD1 is an inhibitory member of theCD28 family of receptors that also includes CD28, CTLA-4, ICOS, andBTLA. PD-1 is expressed on activated B cells, T-cells and myeloid cells(Agata et al. 1996 Int. Immunol 8:765-75). Two ligands for PD1, PD-L1and PD-L2 have been shown to downregulate T-cell activation upon bindingto PD1 (Freeman et al. 2000 J Exp Med 192:1027-34; Latchman et al. 2001Nat Immunol 2:261-8; Carter et al. 2002 Eur J Immunol 32:634-43). PD-L1is abundant in human cancers (Dong et al. 2003 J Mol Med 81:281-7; Blanket al. 2005 Cancer Immunol. Immunother 54:307-314; Konishi et al. 2004Clin Cancer Res 10:5094). Immune suppression can be reversed byinhibiting the local interaction of PD1 with PD-L1.

In one embodiment, the agent comprises the extracellular domain (ECD) ofan inhibitory molecule, e.g., Programmed Death 1 (PD1) can be fused toatransmembrane domain and optionally an intracellular signaling domainsuch as 41BB and CD3 zeta (also referred to herein as a PD1 TFP). In oneembodiment, the PD1 TFP, when used in combinations with ananti-mesothelin TFP described herein, improves the persistence of theT-cell. In one embodiment, the TFP is a PD1 TFP comprising theextracellular domain of PD 1. Alternatively, provided are TFPscontaining an antibody or antibody fragment such as a scFv thatspecifically binds to the Programmed Death-Ligand 1 (PD-L1) orProgrammed Death-Ligand 2 (PD-L2).

In another aspect, the present invention provides a population ofTFP-expressing T-cells, e.g., TFP-T-cells. In some embodiments, thepopulation of TFP-expressing T-cells comprises a mixture of cellsexpressing different TFPs. For example, in one embodiment, thepopulation of TFP-T-cells can include a first cell expressing a TFPhaving an anti-mesothelin binding domain described herein, and a secondcell expressing a TFP having a different anti-mesothelin binding domain,e.g., an anti-mesothelin binding domain described herein that differsfrom the anti-mesothelin binding domain in the TFP expressed by thefirst cell. As another example, the population of TFP-expressing cellscan include a first cell expressing a TFP that includes ananti-mesothelin binding domain, e.g., as described herein, and a secondcell expressing a TFP that includes an antigen binding domain to atarget other than mesothelin (e.g., another tumor-associated antigen).

In another aspect, the present invention provides a population of cellswherein at least one cell in the population expresses a TFP having ananti-mesothelin domain described herein, and a second cell expressinganother agent, e.g., an agent which enhances the activity of aTFP-expressing cell. For example, in one embodiment, the agent can be anagent which inhibits an inhibitory molecule. Inhibitory molecules, e.g.,can, in some embodiments, decrease the ability of a TFP-expressing cellto mount an immune effector response. Examples of inhibitory moleculesinclude PD1, PD-L1, PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1,CD160, 2B4 and TGFR beta. In one embodiment, the agent that inhibits aninhibitory molecule comprises a first polypeptide, e.g., an inhibitorymolecule, associated with a second polypeptide that provides a positivesignal to the cell, e.g., an intracellular signaling domain describedherein.

Disclosed herein are methods for producing in vitro transcribed RNAencoding TFPs. The present invention also includes a TFP encoding RNAconstruct that can be directly transfected into a cell. A method forgenerating mRNA for use in transfection can involve in vitrotranscription (IVT) of a template with specially designed primers,followed by polyA addition, to produce a construct containing 3′ and 5′untranslated sequence (“UTR”), a 5′ cap and/or Internal Ribosome EntrySite (IRES), the nucleic acid to be expressed, and a polyA tail,typically 50-2000 bases in length. RNA so produced can efficientlytransfect different kinds of cells. In one aspect, the template includessequences for the TFP.

In one aspect, the anti-mesothelin TFP is encoded by a messenger RNA(mRNA). In one aspect the mRNA encoding the anti-mesothelin TFP isintroduced into a T-cell for production of a TFP-T-cell. In oneembodiment, the in vitro transcribed RNA TFP can be introduced to a cellas a form of transient transfection. The RNA is produced by in vitrotranscription using a polymerase chain reaction (PCR)-generatedtemplate. DNA of interest from any source can be directly converted byPCR into a template for in vitro mRNA synthesis using appropriateprimers and RNA polymerase. The source of the DNA can be, for example,genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or anyother appropriate source of DNA. The desired template for in vitrotranscription is a TFP of the present invention. In one embodiment, theDNA to be used for PCR contains an open reading frame. The DNA can befrom a naturally occurring DNA sequence from the genome of an organism.In one embodiment, the nucleic acid can include some or all of the 5′and/or 3′ untranslated regions (UTRs). The nucleic acid can includeexons and introns. In one embodiment, the DNA to be used for PCR is ahuman nucleic acid sequence. In another embodiment, the DNA to be usedfor PCR is a human nucleic acid sequence including the 5′ and 3′ UTRs.The DNA can alternatively be an artificial DNA sequence that is notnormally expressed in a naturally occurring organism. An exemplaryartificial DNA sequence is one that contains portions of genes that areligated together to form an open reading frame that encodes a fusionprotein. The portions of DNA that are ligated together can be from asingle organism or from more than one organism.

PCR is used to generate a template for in vitro transcription of mRNAwhich is used for transfection. Methods for performing PCR are wellknown in the art. Primers for use in PCR are designed to have regionsthat are substantially complementary to regions of the DNA to be used asa template for the PCR. “Substantially complementary,” as used herein,refers to sequences of nucleotides where a majority or all of the basesin the primer sequence are complementary, or one or more bases arenon-complementary, or mismatched. Substantially complementary sequencesare able to anneal or hybridize with the intended DNA target underannealing conditions used for PCR. The primers can be designed to besubstantially complementary to any portion of the DNA template. Forexample, the primers can be designed to amplify the portion of a nucleicacid that is normally transcribed in cells (the open reading frame),including 5′ and 3′ UTRs. The primers can also be designed to amplify aportion of a nucleic acid that encodes a particular domain of interest.In one embodiment, the primers are designed to amplify the coding regionof a human cDNA, including all or portions of the 5′ and 3′ UTRs.Primers useful for PCR can be generated by synthetic methods that arewell known in the art. “Forward primers” are primers that contain aregion of nucleotides that are substantially complementary tonucleotides on the DNA template that are upstream of the DNA sequencethat is to be amplified. “Upstream” is used herein to refer to alocation 5, to the DNA sequence to be amplified relative to the codingstrand. “Reverse primers” are primers that contain a region ofnucleotides that are substantially complementary to a double-strandedDNA template that are downstream of the DNA sequence that is to beamplified. “Downstream” is used herein to refer to a location 3′ to theDNA sequence to be amplified relative to the coding strand.

Any DNA polymerase useful for PCR can be used in the methods disclosedherein. The reagents and polymerase are commercially available from anumber of sources.

Chemical structures with the ability to promote stability and/ortranslation efficiency may also be used. The RNA preferably has 5′ and3′ UTRs. In one embodiment, the 5′ UTR is between one and 3,000nucleotides in length. The length of 5′ and 3′ UTR sequences to be addedto the coding region can be altered by different methods, including, butnot limited to, designing primers for PCR that anneal to differentregions of the UTRs. Using this approach, one of ordinary skill in theart can modify the 5′ and 3′ UTR lengths required to achieve optimaltranslation efficiency following transfection of the transcribed RNA.

The 5′ and 3′ UTRs can be the naturally occurring, endogenous 5′ and 3′UTRs for the nucleic acid of interest. Alternatively, UTR sequences thatare not endogenous to the nucleic acid of interest can be added byincorporating the UTR sequences into the forward and reverse primers orby any other modifications of the template. The use of UTR sequencesthat are not endogenous to the nucleic acid of interest can be usefulfor modifying the stability and/or translation efficiency of the RNA.For example, it is known that AU-rich elements in 3′UTR sequences candecrease the stability of mRNA. Therefore, 3′ UTRs can be selected ordesigned to increase the stability of the transcribed RNA based onproperties of UTRs that are well known in the art.

In one embodiment, the 5′ UTR can contain the Kozak sequence of theendogenous nucleic acid. Alternatively, when a 5′ UTR that is notendogenous to the nucleic acid of interest is being added by PCR asdescribed above, a consensus Kozak sequence can be redesigned by addingthe 5′ UTR sequence. Kozak sequences can increase the efficiency oftranslation of some RNA transcripts, but does not appear to be requiredfor all RNAs to enable efficient translation. The requirement for Kozaksequences for many mRNAs is known in the art. In other embodiments the5′ UTR can be 5′UTR of an RNA virus whose RNA genome is stable in cells.In other embodiments various nucleotide analogues can be used in the 3′or 5′ UTR to impede exonuclease degradation of the mRNA.

To enable synthesis of RNA from a DNA template without the need for genecloning, a promoter of transcription should be attached to the DNAtemplate upstream of the sequence to be transcribed. When a sequencethat functions as a promoter for an RNA polymerase is added to the 5′end of the forward primer, the RNA polymerase promoter becomesincorporated into the PCR product upstream of the open reading framethat is to be transcribed. In one preferred embodiment, the promoter isa T7 polymerase promoter, as described elsewhere herein. Other usefulpromoters include, but are not limited to, T3 and SP6 RNA polymerasepromoters. Consensus nucleotide sequences for T7, T3 and SP6 promotersare known in the art.

In a preferred embodiment, the mRNA has both a cap on the 5′ end and a3′ poly(A) tail which determine ribosome binding, initiation oftranslation and stability mRNA in the cell. On a circular DNA template,for instance, plasmid DNA, RNA polymerase produces a long concatamericproduct which is not suitable for expression in eukaryotic cells. Thetranscription of plasmid DNA linearized at the end of the 3′ UTR resultsin normal sized mRNA which is not effective in eukaryotic transfectioneven if it is polyadenylated after transcription.

On a linear DNA template, phage T7 RNA polymerase can extend the 3′ endof the transcript beyond the last base of the template (Schenborn andMierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva andBerzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

The conventional method of integration of polyA/T stretches into a DNAtemplate is molecular cloning. However polyA/T sequence integrated intoplasmid DNA can cause plasmid instability, which is why plasmid DNAtemplates obtained from bacterial cells are often highly contaminatedwith deletions and other aberrations. This makes cloning procedures notonly laborious and time consuming but often not reliable. That is why amethod which allows construction of DNA templates with polyA/T 3′stretch without cloning highly desirable.

The polyA/T segment of the transcriptional DNA template can be producedduring PCR by using a reverse primer containing a polyT tail, such as100 T tail (size can be 50-5000 Ts), or after PCR by any other method,including, but not limited to, DNA ligation or in vitro recombination.Poly(A) tails also provide stability to RNAs and reduce theirdegradation. Generally, the length of a poly(A) tail positivelycorrelates with the stability of the transcribed RNA. In one embodiment,the poly(A) tail is between 100 and 5000 adenosines.

Poly(A) tails of RNAs can be further extended following in vitrotranscription with the use of a poly(A) polymerase, such as E. colipolyA polymerase (E-PAP). In one embodiment, increasing the length of apoly(A) tail from 100 nucleotides to between 300 and 400 nucleotidesresults in about a two-fold increase in the translation efficiency ofthe RNA. Additionally, the attachment of different chemical groups tothe 3′ end can increase mRNA stability. Such attachment can containmodified/artificial nucleotides, aptamers and other compounds. Forexample, ATP analogs can be incorporated into the poly(A) tail usingpoly(A) polymerase. ATP analogs can further increase the stability ofthe RNA.

5′ caps on also provide stability to RNA molecules. In a preferredembodiment, RNAs produced by the methods disclosed herein include a 5′cap. The 5′ cap is provided using techniques known in the art anddescribed herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444(2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al.,Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

The RNAs produced by the methods disclosed herein can also contain aninternal ribosome entry site (IRES) sequence. The IRES sequence may beany viral, chromosomal or artificially designed sequence which initiatescap-independent ribosome binding to mRNA and facilitates the initiationof translation. Any solutes suitable for cell electroporation, which cancontain factors facilitating cellular permeability and viability such assugars, peptides, lipids, proteins, antioxidants, and surfactants can beincluded.

RNA can be introduced into target cells using any of a number ofdifferent methods, for instance, commercially available methods whichinclude, but are not limited to, electroporation (Amaxa Nucleofector-II(Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (HarvardInstruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver,Colo.), Multiporator (Eppendort, Hamburg Germany), cationic liposomemediated transfection using lipofection, polymer encapsulation, peptidemediated transfection, or biolistic particle delivery systems such as“gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther.,12(8):861-70 (2001).

Nucleic Acid Constructs Encoding a TFP

The present invention also provides nucleic acid molecules encoding oneor more TFP constructs described herein. In one aspect, the nucleic acidmolecule is provided as a messenger RNA transcript. In one aspect, thenucleic acid molecule is provided as a DNA construct.

The nucleic acid sequences coding for the desired molecules can beobtained using recombinant methods known in the art, such as, forexample by screening libraries from cells expressing the gene, byderiving the gene from a vector known to include the same, or byisolating directly from cells and tissues containing the same, usingstandard techniques. Alternatively, the gene of interest can be producedsynthetically, rather than cloned.

The present invention also provides vectors in which a DNA of thepresent invention is inserted. Vectors derived from retroviruses such asthe lentivirus are suitable tools to achieve long-term gene transfersince they allow long-term, stable integration of a transgene and itspropagation in daughter cells. Lentiviral vectors have the addedadvantage over vectors derived from onco-retroviruses such as murineleukemia viruses in that they can transduce non-proliferating cells,such as hepatocytes. They also have the added advantage of lowimmunogenicity.

In another embodiment, the vector comprising the nucleic acid encodingthe desired TFP of the invention is an adenoviral vector (A5/35). Inanother embodiment, the expression of nucleic acids encoding TFPs can beaccomplished using of transposons such as sleeping beauty, crisper,CAS9, and zinc finger nucleases (See, June et al. 2009 Nature ReviewsImmunol. 9.10: 704-716, incorporated herein by reference).

The expression constructs of the present invention may also be used fornucleic acid immunization and gene therapy, using standard gene deliveryprotocols. Methods for gene delivery are known in the art (see, e.g.,U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated byreference herein in their entireties). In another embodiment, theinvention provides a gene therapy vector.

The nucleic acid can be cloned into a number of types of vectors. Forexample, the nucleic acid can be cloned into a vector including, but notlimited to a plasmid, a phagemid, a phage derivative, an animal virus,and a cosmid. Vectors of particular interest include expression vectors,replication vectors, probe generation vectors, and sequencing vectors.

Further, the expression vector may be provided to a cell in the form ofa viral vector. Viral vector technology is well known in the art and isdescribed, e.g., in Sambrook et al., 2012, Molecular Cloning: ALaboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY), and inother virology and molecular biology manuals. Viruses, which are usefulas vectors include, but are not limited to, retroviruses, adenoviruses,adeno-associated viruses, herpes viruses, and lentiviruses. In general,a suitable vector contains an origin of replication functional in atleast one organism, a promoter sequence, convenient restrictionendonuclease sites, and one or more selectable markers (e.g., WO1/96584; WO 1/29058; and U.S. Pat. No. 6,326,193).

A number of virally based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. A selected gene can be inserted intoa vector and packaged in retroviral particles using techniques known inthe art. The recombinant virus can then be isolated and delivered tocells of the subject either in vivo or ex vivo. A number of retroviralsystems are known in the art. In some embodiments, adenovirus vectorsare used. A number of adenovirus vectors are known in the art. In oneembodiment, lentivirus vectors are used.

Additional promoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave been shown to contain functional elements downstream of the startsite as well. The spacing between promoter elements frequently isflexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline. Depending on the promoter,it appears that individual elements can function either cooperatively orindependently to activate transcription.

An example of a promoter that is capable of expressing a TFP transgenein a mammalian T-cell is the EF1a promoter. The native EF1a promoterdrives expression of the alpha subunit of the elongation factor-1complex, which is responsible for the enzymatic delivery of aminoacyltRNAs to the ribosome. The EF1a promoter has been extensively used inmammalian expression plasmids and has been shown to be effective indriving TFP expression from transgenes cloned into a lentiviral vector(see, e.g., Milone et al., Mol. Ther. 17(8): 1453-1464 (2009)). Anotherexample of a promoter is the immediate early cytomegalovirus (CMV)promoter sequence. This promoter sequence is a strong constitutivepromoter sequence capable of driving high levels of expression of anypolynucleotide sequence operatively linked thereto. However, otherconstitutive promoter sequences may also be used, including, but notlimited to the simian virus 40 (SV40) early promoter, mouse mammarytumor virus (MMTV), human immunodeficiency virus (HIV) long terminalrepeat (LTR) promoter, MoMuLV promoter, an avian leukemia viruspromoter, an Epstein-Barr virus immediate early promoter, a Rous sarcomavirus promoter, as well as human gene promoters such as, but not limitedto, the actin promoter, the myosin promoter, the elongation factor-1apromoter, the hemoglobin promoter, and the creatine kinase promoter.Further, the invention should not be limited to the use of constitutivepromoters. Inducible promoters are also contemplated as part of theinvention. The use of an inducible promoter provides a molecular switchcapable of turning on expression of the polynucleotide sequence which itis operatively linked when such expression is desired, or turning offthe expression when expression is not desired. Examples of induciblepromoters include, but are not limited to a metallothionine promoter, aglucocorticoid promoter, a progesterone promoter, and atetracycline-regulated promoter.

In order to assess the expression of a TFP polypeptide or portionsthereof, the expression vector to be introduced into a cell can alsocontain either a selectable marker gene or a reporter gene or both tofacilitate identification and selection of expressing cells from thepopulation of cells sought to be transfected or infected through viralvectors. In other aspects, the selectable marker may be carried on aseparate piece of DNA and used in a co-transfection procedure. Bothselectable markers and reporter genes may be flanked with appropriateregulatory sequences to enable expression in the host cells. Usefulselectable markers include, for example, antibiotic-resistance genes,such as neo and the like.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient organism or tissue and that encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assayed at asuitable time after the DNA has been introduced into the recipientcells. Suitable reporter genes may include genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the green fluorescent protein gene (e.g.,Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expressionsystems are well known and may be prepared using known techniques orobtained commercially. In general, the construct with the minimal 5′flanking region showing the highest level of expression of reporter geneis identified as the promoter. Such promoter regions may be linked to areporter gene and used to evaluate agents for the ability to modulatepromoter-driven transcription.

Methods of introducing and expressing genes into a cell are known in theart. In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast, orinsect cell by any method in the art. For example, the expression vectorcan be transferred into a host cell by physical, chemical, or biologicalmeans.

Physical methods for introducing a polynucleotide into a host cellinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art (see, e.g., Sambrook et al., 2012, MolecularCloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press,NY). One method for the introduction of a polynucleotide into a hostcell is calcium phosphate transfection

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virusI, adenoviruses and adeno-associated viruses, and the like (see, e.g.,U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle). Other methodsof state-of-the-art targeted delivery of nucleic acids are available,such as delivery of polynucleotides with targeted nanoparticles or othersuitable sub-micron sized delivery system.

In the case where a non-viral delivery system is utilized, an exemplarydelivery vehicle is a liposome. The use of lipid formulations iscontemplated for the introduction of the nucleic acids into a host cell(in vitro, ex vivo or in vivo). In another aspect, the nucleic acid maybe associated with a lipid. The nucleic acid associated with a lipid maybe encapsulated in the aqueous interior of a liposome, interspersedwithin the lipid bilayer of a liposome, attached to a liposome via alinking molecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid,lipid/DNA or lipid/expression vector associated compositions are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in size or shape. Lipids arefatty substances which may be naturally occurring or synthetic lipids.For example, lipids include the fatty droplets that naturally occur inthe cytoplasm as well as the class of compounds which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. Forexample, dimyristyl phosphatidylcholine (“DMPC”) can be obtained fromSigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K& K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtainedfrom Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) andother lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform or chloroform/methanolcan be stored at about −20° C. Chloroform is used as the only solventsince it is more readily evaporated than methanol. “Liposome” is ageneric term encompassing a variety of single and multilamellar lipidvehicles formed by the generation of enclosed lipid bilayers oraggregates. Liposomes can be characterized as having vesicularstructures with a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh et al.,1991 Glycobiology 5: 505-10). However, compositions that have differentstructures in solution than the normal vesicular structure are alsoencompassed. For example, the lipids may assume a micellar structure ormerely exist as nonuniform aggregates of lipid molecules. Alsocontemplated are lipofectamine-nucleic acid complexes.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell or otherwise expose a cell to the inhibitor of the presentinvention, in order to confirm the presence of the recombinant DNAsequence in the host cell, a variety of assays may be performed. Suchassays include, for example, “molecular biological” assays well known tothose of skill in the art, such as Southern and Northern blotting,RT-PCR and PCR; “biochemical” assays, such as detecting the presence orabsence of a particular peptide, e.g., by immunological means (ELISAsand Western blots) or by assays described herein to identify agentsfalling within the scope of the invention.

The present invention further provides a vector comprising a TFPencoding nucleic acid molecule. In one aspect, a TFP vector can bedirectly transduced into a cell, e.g., a T-cell. In one aspect, thevector is a cloning or expression vector, e.g., a vector including, butnot limited to, one or more plasmids (e.g., expression plasmids, cloningvectors, minicircles, minivectors, double minute chromosomes),retroviral and lentiviral vector constructs. In one aspect, the vectoris capable of expressing the TFP construct in mammalian T-cells. In oneaspect, the mammalian T-cell is a human T-cell.

Sources of T-Cells

Prior to expansion and genetic modification, a source of T-cells isobtained from a subject. The term “subject” is intended to includeliving organisms in which an immune response can be elicited (e.g.,mammals). Examples of subjects include humans, dogs, cats, mice, rats,and transgenic species thereof. T-cells can be obtained from a number ofsources, including peripheral blood mononuclear cells, bone marrow,lymph node tissue, cord blood, thymus tissue, tissue from a site ofinfection, ascites, pleural effusion, spleen tissue, and tumors. Incertain aspects of the present invention, any number of T-cell linesavailable in the art, may be used. In certain aspects of the presentinvention, T-cells can be obtained from a unit of blood collected from asubject using any number of techniques known to the skilled artisan,such as Ficoll™ separation. In one preferred aspect, cells from thecirculating blood of an individual are obtained by apheresis. Theapheresis product typically contains lymphocytes, including T-cells,monocytes, granulocytes, B cells, other nucleated white blood cells, redblood cells, and platelets. In one aspect, the cells collected byapheresis may be washed to remove the plasma fraction and to place thecells in an appropriate buffer or media for subsequent processing steps.In one aspect of the invention, the cells are washed with phosphatebuffered saline (PBS). In an alternative aspect, the wash solution lackscalcium and may lack magnesium or may lack many if not all divalentcations. Initial activation steps in the absence of calcium can lead tomagnified activation. As those of ordinary skill in the art wouldreadily appreciate a washing step may be accomplished by methods knownto those in the art, such as by using a semi-automated “flow-through”centrifuge (for example, the Cobe 2991 cell processor, the BaxterCytoMate, or the Haemonetics Cell Saver 5) according to themanufacturer's instructions. After washing, the cells may be resuspendedin a variety of biocompatible buffers, such as, for example, Ca-free,Mg-free PBS, PlasmaLyte A, or other saline solution with or withoutbuffer. Alternatively, the undesirable components of the apheresissample may be removed and the cells directly resuspended in culturemedia.

In one aspect, T-cells are isolated from peripheral blood lymphocytes bylysing the red blood cells and depleting the monocytes, for example, bycentrifugation through a PERCOLL™ gradient or by counterflow centrifugalelutriation. A specific subpopulation of T-cells, such as CD3+, CD28+,CD4+, CD8+, CD45RA+, and CD45RO+ T-cells, can be further isolated bypositive or negative selection techniques. For example, in one aspect,T-cells are isolated by incubation with anti-CD3/anti-CD28 (e.g.,3×28)-conjugated beads, such as DYNABEADS™ M-450 CD3/CD28 T, for a timeperiod sufficient for positive selection of the desired T-cells. In oneaspect, the time period is about 30 minutes. In a further aspect, thetime period ranges from 30 minutes to 36 hours or longer and all integervalues there between. In a further aspect, the time period is at least1, 2, 3, 4, 5, or 6 hours. In yet another preferred aspect, the timeperiod is 10 to 24 hours. In one aspect, the incubation time period is24 hours. Longer incubation times may be used to isolate T-cells in anysituation where there are few T-cells as compared to other cell types,such in isolating tumor infiltrating lymphocytes (TIL) from tumor tissueor from immunocompromised individuals. Further, use of longer incubationtimes can increase the efficiency of capture of CD8+ T-cells. Thus, bysimply shortening or lengthening the time T-cells are allowed to bind tothe CD3/CD28 beads and/or by increasing or decreasing the ratio of beadsto T-cells (as described further herein), subpopulations of T-cells canbe preferentially selected for or against at culture initiation or atother time points during the process. Additionally, by increasing ordecreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on thebeads or other surface, subpopulations of T-cells can be preferentiallyselected for or against at culture initiation or at other desired timepoints. The skilled artisan would recognize that multiple rounds ofselection can also be used in the context of this invention. In certainaspects, it may be desirable to perform the selection procedure and usethe “unselected” cells in the activation and expansion process.“Unselected” cells can also be subjected to further rounds of selection.

Enrichment of a T-cell population by negative selection can beaccomplished with a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. One method is cellsorting and/or selection via negative magnetic immunoadherence or flowcytometry that uses a cocktail of monoclonal antibodies directed to cellsurface markers present on the cells negatively selected. For example,to enrich for CD4+ cells by negative selection, a monoclonal antibodycocktail typically includes antibodies to CD14, CD20, CD11b, CD16,HLA-DR, and CD8. In certain aspects, it may be desirable to enrich foror positively select for regulatory T-cells which typically expressCD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, in certainaspects, T regulatory cells are depleted by anti-C25 conjugated beads orother similar method of selection.

In one embodiment, a T-cell population can be selected that expressesone or more of IFN-γ, TNF-alpha, IL-17A, IL-2, IL-3, IL-4, GM-CSF,IL-10, IL-13, granzyme B, and perforin, or other appropriate molecules,e.g., other cytokines. Methods for screening for cell expression can bedetermined, e.g., by the methods described in PCT Publication No.: WO2013/126712.

For isolation of a desired population of cells by positive or negativeselection, the concentration of cells and surface (e.g., particles suchas beads) can be varied. In certain aspects, it may be desirable tosignificantly decrease the volume in which beads and cells are mixedtogether (e.g., increase the concentration of cells), to ensure maximumcontact of cells and beads. For example, in one aspect, a concentrationof 2 billion cells/mL is used. In one aspect, a concentration of 1billion cells/mL is used. In a further aspect, greater than 100 millioncells/mL is used. In a further aspect, a concentration of cells of 10,15, 20, 25, 30, 35, 40, 45, or 50 million cells/mL is used. In yet oneaspect, a concentration of cells from 75, 80, 85, 90, 95, or 100 millioncells/mL is used. In further aspects, concentrations of 125 or 150million cells/mL can be used. Using high concentrations can result inincreased cell yield, cell activation, and cell expansion. Further, useof high cell concentrations allows more efficient capture of cells thatmay weakly express target antigens of interest, such as CD28-negativeT-cells, or from samples where there are many tumor cells present (e.g.,leukemic blood, tumor tissue, etc.). Such populations of cells may havetherapeutic value and would be desirable to obtain. For example, usinghigh concentration of cells allows more efficient selection of CD8+T-cells that normally have weaker CD28 expression.

In a related aspect, it may be desirable to use lower concentrations ofcells. By significantly diluting the mixture of T-cells and surface(e.g., particles such as beads), interactions between the particles andcells is minimized. This selects for cells that express high amounts ofdesired antigens to be bound to the particles. For example, CD4+ T-cellsexpress higher levels of CD28 and are more efficiently captured thanCD8+ T-cells in dilute concentrations. In one aspect, the concentrationof cells used is 5×10⁶/mL. In other aspects, the concentration used canbe from about 1×10⁵/mL to 1×10⁶/mL, and any integer value in between. Inother aspects, the cells may be incubated on a rotator for varyinglengths of time at varying speeds at either 2-10° C. or at roomtemperature.

T-cells for stimulation can also be frozen after a washing step. Wishingnot to be bound by theory, the freeze and subsequent thaw step providesa more uniform product by removing granulocytes and to some extentmonocytes in the cell population. After the washing step that removesplasma and platelets, the cells may be suspended in a freezing solution.While many freezing solutions and parameters are known in the art andwill be useful in this context, one method involves using PBS containing20% DMSO and 8% human serum albumin, or culture media containing 10%Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitablecell freezing media containing for example, Hespan and PlasmaLyte A, thecells then are frozen to −80° C. at a rate of 1 per minute and stored inthe vapor phase of a liquid nitrogen storage tank. Other methods ofcontrolled freezing may be used as well as uncontrolled freezingimmediately at −20° C. or in liquid nitrogen. In certain aspects,cryopreserved cells are thawed and washed as described herein andallowed to rest for one hour at room temperature prior to activationusing the methods of the present invention.

Also contemplated in the context of the invention is the collection ofblood samples or apheresis product from a subject at a time period priorto when the expanded cells as described herein might be needed. As such,the source of the cells to be expanded can be collected at any timepoint necessary, and desired cells, such as T-cells, isolated and frozenfor later use in T-cell therapy for any number of diseases or conditionsthat would benefit from T-cell therapy, such as those described herein.In one aspect a blood sample or an apheresis is taken from a generallyhealthy subject. In certain aspects, a blood sample or an apheresis istaken from a generally healthy subject who is at risk of developing adisease, but who has not yet developed a disease, and the cells ofinterest are isolated and frozen for later use. In certain aspects, theT-cells may be expanded, frozen, and used at a later time. In certainaspects, samples are collected from a patient shortly after diagnosis ofa particular disease as described herein but prior to any treatments. Ina further aspect, the cells are isolated from a blood sample or anapheresis from a subject prior to any number of relevant treatmentmodalities, including but not limited to treatment with agents such asnatalizumab, efalizumab, antiviral agents, chemotherapy, radiation,immunosuppressive agents, such as cyclosporin, azathioprine,methotrexate, mycophenolate, and FK506, antibodies, or otherimmunoablative agents such as alemtuzumab, anti-CD3 antibodies, cytoxan,fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids,FR901228, and irradiation.

In a further aspect of the present invention, T-cells are obtained froma patient directly following treatment that leaves the subject withfunctional T-cells. In this regard, it has been observed that followingcertain cancer treatments, in particular treatments with drugs thatdamage the immune system, shortly after treatment during the period whenpatients would normally be recovering from the treatment, the quality ofT-cells obtained may be optimal or improved for their ability to expandex vivo. Likewise, following ex vivo manipulation using the methodsdescribed herein, these cells may be in a preferred state for enhancedengraftment and in vivo expansion. Thus, it is contemplated within thecontext of the present invention to collect blood cells, includingT-cells, dendritic cells, or other cells of the hematopoietic lineage,during this recovery phase. Further, in certain aspects, mobilization(for example, mobilization with GM-CSF) and conditioning regimens can beused to create a condition in a subject wherein repopulation,recirculation, regeneration, and/or expansion of particular cell typesis favored, especially during a defined window of time followingtherapy. Illustrative cell types include T-cells, B cells, dendriticcells, and other cells of the immune system.

Activation and Expansion of T Cells

T-cells may be activated and expanded generally using methods asdescribed, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055;6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575;7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874;6,797,514; 6,867,041, and 7,572,631.

Generally, the T-cells of the invention may be expanded by contact witha surface having attached thereto an agent that stimulates a CD3/TCRcomplex associated signal and a ligand that stimulates a costimulatorymolecule on the surface of the T-cells. In particular, T-cellpopulations may be stimulated as described herein, such as by contactwith an anti-CD3 antibody, or antigen-binding fragment thereof, or ananti-CD2 antibody immobilized on a surface, or by contact with a proteinkinase C activator (e.g., bryostatin) in conjunction with a calciumionophore. For co-stimulation of an accessory molecule on the surface ofthe T-cells, a ligand that binds the accessory molecule is used. Forexample, a population of T-cells can be contacted with an anti-CD3antibody and an anti-CD28 antibody, under conditions appropriate forstimulating proliferation of the T-cells. To stimulate proliferation ofeither CD4+ T-cells or CD8+ T-cells, an anti-CD3 antibody and ananti-CD28 antibody. Examples of an anti-CD28 antibody include 9.3, B-T3,XR-CD28 (Diaclone, Besancon, France) can be used as can other methodscommonly known in the art (Berg et al., Transplant Proc.30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328,1999; Garland et al., J. Immunol. Meth. 227(1-2):53-63, 1999).

T-cells that have been exposed to varied stimulation times may exhibitdifferent characteristics. For example, typical blood or apheresedperipheral blood mononuclear cell products have a helper T-cellpopulation (TH, CD4+) that is greater than the cytotoxic or suppressorT-cell population (TC, CD8+). Ex vivo expansion of T-cells bystimulating CD3 and CD28 receptors produces a population of T-cells thatprior to about days 8-9 consists predominately of TH cells, while afterabout days 8-9, the population of T-cells comprises an increasinglygreater population of TC cells. Accordingly, depending on the purpose oftreatment, infusing a subject with a T-cell population comprisingpredominately of TH cells may be advantageous. Similarly, if anantigen-specific subset of TC cells has been isolated it may bebeneficial to expand this subset to a greater degree.

Further, in addition to CD4 and CD8 markers, other phenotypic markersvary significantly, but in large part, reproducibly during the course ofthe cell expansion process. Thus, such reproducibility enables theability to tailor an activated T-cell product for specific purposes.

Once an anti-mesothelin TFP is constructed, various assays can be usedto evaluate the activity of the molecule, such as but not limited to,the ability to expand T-cells following antigen stimulation, sustainT-cell expansion in the absence of re-stimulation, and anti-canceractivities in appropriate in vitro and animal models. Assays to evaluatethe effects of an anti-mesothelin TFP are described in further detailbelow

Western blot analysis of TFP expression in primary T-cells can be usedto detect the presence of monomers and dimers (see, e.g., Milone et al.,Molecular Therapy 17(8): 1453-1464 (2009)). Very briefly, T-cells (1:1mixture of CD4⁺ and CD8⁺ T-cells) expressing the TFPs are expanded invitro for more than 10 days followed by lysis and SDS-PAGE underreducing conditions. TFPs are detected by Western blotting using anantibody to a TCR chain. The same T-cell subsets are used for SDS-PAGEanalysis under non-reducing conditions to permit evaluation of covalentdimer formation.

In vitro expansion of TFP⁺ T-cells following antigen stimulation can bemeasured by flow cytometry. For example, a mixture of CD4⁺ and CD8⁺T-cells are stimulated with alphaCD3/alphaCD28 and APCs followed bytransduction with lentiviral vectors expressing GFP under the control ofthe promoters to be analyzed. Exemplary promoters include the CMV IEgene, EF-lalpha, ubiquitin C, or phosphoglycerokinase (PGK) promoters.GFP fluorescence is evaluated on day 6 of culture in the CD4+ and/orCD8+ T-cell subsets by flow cytometry (see, e.g., Milone et al.,Molecular Therapy 17(8): 1453-1464 (2009)). Alternatively, a mixture ofCD4+ and CD8+ T-cells are stimulated with alphaCD3/alphaCD28 coatedmagnetic beads on day 0, and transduced with TFP on day 1 using abicistronic lentiviral vector expressing TFP along with eGFP using a 2Aribosomal skipping sequence. Cultures are re-stimulated with eithermesothelin+K562 cells (K562-mesothelin), wild-type K562 cells (K562 wildtype) or K562 cells expressing hCD32 and 4-1BBL in the presence ofantiCD3 and anti-CD28 antibody (K562-BBL-3/28) following washing.Exogenous IL-2 is added to the cultures every other day at 100 IU/mL.GFP+ T-cells are enumerated by flow cytometry using bead-based counting(see, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)).

Sustained TFP+ T-cell expansion in the absence of re-stimulation canalso be measured (see, e.g., Milone et al., Molecular Therapy 17(8):1453-1464 (2009)). Briefly, mean T-cell volume (fl) is measured on day 8of culture using a Coulter Multisizer III particle counter followingstimulation with alphaCD3/alphaCD28 coated magnetic beads on day 0, andtransduction with the indicated TFP on day 1.

Animal models can also be used to measure a TFP-T activity. For example,xenograft model using human mesothelin-specific TFP+ T-cells to treat acancer in immunodeficient mice (see, e.g., Milone et al., MolecularTherapy 17(8): 1453-1464 (2009)). Very briefly, after establishment ofcancer, mice are randomized as to treatment groups. Different numbers ofengineered T-cells are coinjected at a 1:1 ratio into NOD/SCID/γ−/− micebearing cancer. The number of copies of each vector in spleen DNA frommice is evaluated at various times following T-cell injection. Animalsare assessed for cancer at weekly intervals. Peripheral bloodmesothelin+ cancer cell counts are measured in mice that are injectedwith alphamesothelin-zeta TFP+ T-cells or mock-transduced T-cells.Survival curves for the groups are compared using the log-rank test. Inaddition, absolute peripheral blood CD4+ and CD8+ T-cell counts 4 weeksfollowing T-cell injection in NOD/SCID/γ−/−mice can also be analyzed.Mice are injected with cancer cells and 3 weeks later are injected withT-cells engineered to express TFP by a bicistronic lentiviral vectorthat encodes the TFP linked to eGFP. T-cells are normalized to 45-50%input GFP+ T-cells by mixing with mock-transduced cells prior toinjection, and confirmed by flow cytometry. Animals are assessed forcancer at 1-week intervals. Survival curves for the TFP+ T-cell groupsare compared using the log-rank test.

Dose dependent TFP treatment response can be evaluated (see, e.g.,Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)). For example,peripheral blood is obtained 35-70 days after establishing cancer inmice injected on day 21 with TFP T-cells, an equivalent number ofmock-transduced T-cells, or no T-cells. Mice from each group arerandomly bled for determination of peripheral blood mesothelin+ cancercell counts and then killed on days 35 and 49. The remaining animals areevaluated on days 57 and 70.

Assessment of cell proliferation and cytokine production has beenpreviously described, e.g., at Milone et al., Molecular Therapy 17(8):1453-1464 (2009). Briefly, assessment of TFP-mediated proliferation isperformed in microtiter plates by mixing washed T-cells with cellsexpressing mesothelin or CD32 and CD137 (KT32-BBL) for a finalT-cell:cell expressing mesothelin ratio of 2:1. Cells expressingmesothelin cells are irradiated with gamma-radiation prior to use.Anti-CD3 (clone OKT3) and anti-CD28 (clone 9.3) monoclonal antibodiesare added to cultures with KT32-BBL cells to serve as a positive controlfor stimulating T-cell proliferation since these signals supportlong-term CD8+ T-cell expansion ex vivo. T-cells are enumerated incultures using CountBright™ fluorescent beads (Invitrogen) and flowcytometry as described by the manufacturer. TFP+ T-cells are identifiedby GFP expression using T-cells that are engineered with eGFP-2A linkedTFP-expressing lentiviral vectors. For TFP+ T-cells not expressing GFP,the TFP+ T-cells are detected with biotinylated recombinant mesothelinprotein and a secondary avidin-PE conjugate. CD4+ and CD8+ expression onT-cells are also simultaneously detected with specific monoclonalantibodies (BD Biosciences). Cytokine measurements are performed onsupernatants collected 24 hours following re-stimulation using the humanTH1/TH2 cytokine cytometric bead array kit (BD Biosciences) accordingthe manufacturer's instructions. Fluorescence is assessed using aFACScalibur flow cytometer, and data is analyzed according to themanufacturer's instructions.

Cytotoxicity can be assessed by a standard ⁵¹Cr-release assay (see,e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)).Briefly, target cells are loaded with ⁵¹Cr (as NaCrO₄, New EnglandNuclear) at 37° C. for 2 hours with frequent agitation, washed twice incomplete RPMI medium and plated into microtiter plates. Effector T-cellsare mixed with target cells in the wells in complete RPMI at varyingratios of effector cell:target cell (E:T). Additional wells containingmedia only (spontaneous release, SR) or a 1% solution of triton-X 100detergent (total release, TR) are also prepared. After 4 hours ofincubation at 37° C., supernatant from each well is harvested. Released⁵¹Cr is then measured using a gamma particle counter (Packard InstrumentCo., Waltham, Mass.). Each condition is performed in at leasttriplicate, and the percentage of lysis is calculated using the formula:% Lysis=(ER-SR)/(TR-SR), where ER represents the average ⁵¹Cr releasedfor each experimental condition.

Imaging technologies can be used to evaluate specific trafficking andproliferation of TFPs in tumor-bearing animal models. Such assays havebeen described, e.g., in Barrett et al., Human Gene Therapy 22:1575-1586(2011). Briefly, NOD/SCID/γc−/− (NSG) mice are injected IV with cancercells followed 7 days later with T-cells 4 hour after electroporationwith the TFP constructs. The T-cells are stably transfected with alentiviral construct to express firefly luciferase, and mice are imagedfor bioluminescence. Alternatively, therapeutic efficacy and specificityof a single injection of TFP+ T-cells in a cancer xenograft model can bemeasured as follows: NSG mice are injected with cancer cells transducedto stably express firefly luciferase, followed by a single tail-veininjection of T-cells electroporated with mesothelin TFP 7 days later.Animals are imaged at various time points post injection. For example,photon-density heat maps of firefly luciferase positive cancer inrepresentative mice at day 5 (2 days before treatment) and day 8 (24hours post TFP+ PBLs) can be generated.

Other assays, including those described in the Example section herein aswell as those that are known in the art can also be used to evaluate theanti-mesothelin TFP constructs of the invention.

Therapeutic Applications

Mesothelin Associated Diseases and/or Disorders

In one aspect, the invention provides methods for treating a diseaseassociated with mesothelin expression. In one aspect, the inventionprovides methods for treating a disease wherein part of the tumor isnegative for mesothelin and part of the tumor is positive formesothelin. For example, the TFP of the invention is useful for treatingsubjects that have undergone treatment for a disease associated withelevated expression of mesothelin, wherein the subject that hasundergone treatment for elevated levels of mesothelin exhibits a diseaseassociated with elevated levels of mesothelin.

In one aspect, the invention pertains to a vector comprisinganti-mesothelin TFP operably linked to promoter for expression inmammalian T-cells. In one aspect, the invention provides a recombinantT-cell expressing the mesothelin TFP for use in treatingmesothelin-expressing tumors, wherein the recombinant T-cell expressingthe mesothelin TFP is termed a mesothelin TFP-T. In one aspect, themesothelin TFP-T of the invention is capable of contacting a tumor cellwith at least one mesothelin TFP of the invention expressed on itssurface such that the TFP-T targets the tumor cell and growth of thetumor is inhibited.

In one aspect, the invention pertains to a method of inhibiting growthof a mesothelin-expressing tumor cell, comprising contacting the tumorcell with a mesothelin TFP T-cell of the present invention such that theTFP-T is activated in response to the antigen and targets the cancercell, wherein the growth of the tumor is inhibited.

In one aspect, the invention pertains to a method of treating cancer ina subject. The method comprises administering to the subject amesothelin TFP T-cell of the present invention such that the cancer istreated in the subject. An example of a cancer that is treatable by themesothelin TFP T-cell of the invention is a cancer associated withexpression of mesothelin. In one aspect, the cancer is a mesothelioma.In one aspect, the cancer is a pancreatic cancer. In one aspect, thecancer is an ovarian cancer. In one aspect, the cancer is a stomachcancer. In one aspect, the cancer is a lung cancer. In one aspect, thecancer is an endometrial cancer. In some embodiments, mesothelin TFPtherapy can be used in combination with one or more additional therapies

The invention includes a type of cellular therapy where T-cells aregenetically modified to express a TFP and the TFP-expressing T-cell isinfused to a recipient in need thereof. The infused cell is able to killtumor cells in the recipient. Unlike antibody therapies, TFP-expressingT-cells are able to replicate in vivo, resulting in long-termpersistence that can lead to sustained tumor control. In variousaspects, the T-cells administered to the patient, or their progeny,persist in the patient for at least one month, two month, three months,four months, five months, six months, seven months, eight months, ninemonths, ten months, eleven months, twelve months, thirteen months,fourteen month, fifteen months, sixteen months, seventeen months,eighteen months, nineteen months, twenty months, twenty-one months,twenty-two months, twenty-three months, two years, three years, fouryears, or five years after administration of the T-cell to the patient.

The invention also includes a type of cellular therapy where T-cells aremodified, e.g., by in vitro transcribed RNA, to transiently express aTFP and the TFP-expressing T-cell is infused to a recipient in needthereof. The infused cell is able to kill tumor cells in the recipient.Thus, in various aspects, the T-cells administered to the patient, ispresent for less than one month, e.g., three weeks, two weeks, or oneweek, after administration of the T-cell to the patient.

Without wishing to be bound by any particular theory, the anti-tumorimmunity response elicited by the TFP-expressing T-cells may be anactive or a passive immune response, or alternatively may be due to adirect vs indirect immune response. In one aspect, the TFP transducedT-cells exhibit specific proinflammatory cytokine secretion and potentcytolytic activity in response to human cancer cells expressing themesothelin antigen, resist soluble mesothelin inhibition, mediatebystander killing and/or mediate regression of an established humantumor. For example, antigen-less tumor cells within a heterogeneousfield of mesothelin-expressing tumor may be susceptible to indirectdestruction by mesothelin-redirected T-cells that has previously reactedagainst adjacent antigen-positive cancer cells.

In one aspect, the human TFP-modified T-cells of the invention may be atype of vaccine for ex vivo immunization and/or in vivo therapy in amammal. In one aspect, the mammal is a human.

With respect to ex vivo immunization, at least one of the followingoccurs in vitro prior to administering the cell into a mammal: i)expansion of the cells, ii) introducing a nucleic acid encoding a TFP tothe cells or iii) cryopreservation of the cells.

Ex vivo procedures are well known in the art and are discussed morefully below. Briefly, cells are isolated from a mammal (e.g., a human)and genetically modified (i.e., transduced or transfected in vitro) witha vector expressing a TFP disclosed herein. The TFP-modified cell can beadministered to a mammalian recipient to provide a therapeutic benefit.The mammalian recipient may be a human and the TFP-modified cell can beautologous with respect to the recipient. Alternatively, the cells canbe allogeneic, syngeneic or xenogeneic with respect to the recipient.

The procedure for ex vivo expansion of hematopoietic stem and progenitorcells is described in U.S. Pat. No. 5,199,942, incorporated herein byreference, can be applied to the cells of the present invention. Othersuitable methods are known in the art, therefore the present inventionis not limited to any particular method of ex vivo expansion of thecells. Briefly, ex vivo culture and expansion of T-cells comprises: (1)collecting CD34+ hematopoietic stem and progenitor cells from a mammalfrom peripheral blood harvest or bone marrow explants; and (2) expandingsuch cells ex vivo. In addition to the cellular growth factors describedin U.S. Pat. No. 5,199,942, other factors such as flt3-L, IL-1, IL-3 andc-kit ligand, can be used for culturing and expansion of the cells.

In addition to using a cell-based vaccine in terms of ex vivoimmunization, the present invention also provides compositions andmethods for in vivo immunization to elicit an immune response directedagainst an antigen in a patient.

Generally, the cells activated and expanded as described herein may beutilized in the treatment and prevention of diseases that arise inindividuals who are immunocompromised. In particular, the TFP-modifiedT-cells of the invention are used in the treatment of diseases,disorders and conditions associated with expression of mesothelin. Incertain aspects, the cells of the invention are used in the treatment ofpatients at risk for developing diseases, disorders and conditionsassociated with expression of mesothelin. Thus, the present inventionprovides methods for the treatment or prevention of diseases, disordersand conditions associated with expression of mesothelin comprisingadministering to a subject in need thereof, a therapeutically effectiveamount of the TFP-modified T-cells of the invention.

In one aspect the TFP-T-cells of the inventions may be used to treat aproliferative disease such as a cancer or malignancy or a precancerouscondition. In one aspect, the cancer is a mesothelioma. In one aspect,the cancer is a pancreatic cancer. In one aspect, the cancer is anovarian cancer. In one aspect, the cancer is a stomach cancer. In oneaspect, the cancer is a lung cancer. In one aspect, the cancer is aendometrial cancer. Further a disease associated with mesothelinexpression includes, but is not limited to, e.g., atypical and/ornon-classical cancers, malignancies, precancerous conditions orproliferative diseases expressing mesothelin. Non-cancer relatedindications associated with expression of mesothelin include, but arenot limited to, e.g., autoimmune disease, (e.g., lupus), inflammatorydisorders (allergy and asthma) and transplantation.

The TFP-modified T-cells of the present invention may be administeredeither alone, or as a pharmaceutical composition in combination withdiluents and/or with other components such as IL-2 or other cytokines orcell populations.

The present invention also provides methods for inhibiting theproliferation or reducing a mesothelin-expressing cell population, themethods comprising contacting a population of cells comprising amesothelin-expressing cell with an anti-mesothelin TFP-T-cell of theinvention that binds to the mesothelin-expressing cell. In a specificaspect, the present invention provides methods for inhibiting theproliferation or reducing the population of cancer cells expressingmesothelin, the methods comprising contacting the mesothelin-expressingcancer cell population with an anti-mesothelin TFP-T-cell of theinvention that binds to the mesothelin-expressing cell. In one aspect,the present invention provides methods for inhibiting the proliferationor reducing the population of cancer cells expressing mesothelin, themethods comprising contacting the mesothelin-expressing cancer cellpopulation with an anti-mesothelin TFP-T-cell of the invention thatbinds to the mesothelin-expressing cell. In certain aspects, theanti-mesothelin TFP-T-cell of the invention reduces the quantity,number, amount or percentage of cells and/or cancer cells by at least25%, at least 30%, at least 40%, at least 50%, at least 65%, at least75%, at least 85%, at least 95%, or at least 99% in a subject with oranimal model a cancer associated with mesothelin-expressing cellsrelative to a negative control. In one aspect, the subject is a human.

The present invention also provides methods for preventing, treatingand/or managing a disease associated with mesothelin-expressing cells(e.g., a cancer expressing mesothelin), the methods comprisingadministering to a subject in need an anti-mesothelin TFP-T-cell of theinvention that binds to the mesothelin-expressing cell. In one aspect,the subject is a human. Non-limiting examples of disorders associatedwith mesothelin-expressing cells include autoimmune disorders (such aslupus), inflammatory disorders (such as allergies and asthma) andcancers (such as pancreatic cancer, ovarian cancer, stomach cancer, lungcancer, or endometrial cancer. or atypical cancers expressingmesothelin).

The present invention also provides methods for preventing, treatingand/or managing a disease associated with mesothelin-expressing cells,the methods comprising administering to a subject in need ananti-mesothelin TFP-T-cell of the invention that binds to themesothelin-expressing cell. In one aspect, the subject is a human.

The present invention provides methods for preventing relapse of cancerassociated with mesothelin-expressing cells, the methods comprisingadministering to a subject in need thereof an anti-mesothelin TFP-T-cellof the invention that binds to the mesothelin-expressing cell. In oneaspect, the methods comprise administering to the subject in needthereof an effective amount of an anti-mesothelin TFP-T-cell describedherein that binds to the mesothelin-bmcaexpressing cell in combinationwith an effective amount of another therapy.

Combination Therapies

A TFP-expressing cell described herein may be used in combination withother known agents and therapies. Administered “in combination”, as usedherein, means that two (or more) different treatments are delivered tothe subject during the course of the subject's affliction with thedisorder, e.g., the two or more treatments are delivered after thesubject has been diagnosed with the disorder and before the disorder hasbeen cured or eliminated or treatment has ceased for other reasons. Insome embodiments, the delivery of one treatment is still occurring whenthe delivery of the second begins, so that there is overlap in terms ofadministration. This is sometimes referred to herein as “simultaneous”or “concurrent delivery”. In other embodiments, the delivery of onetreatment ends before the delivery of the other treatment begins. Insome embodiments of either case, the treatment is more effective becauseof combined administration. For example, the second treatment is moreeffective, e.g., an equivalent effect is seen with less of the secondtreatment, or the second treatment reduces symptoms to a greater extent,than would be seen if the second treatment were administered in theabsence of the first treatment or the analogous situation is seen withthe first treatment. In some embodiments, delivery is such that thereduction in a symptom, or other parameter related to the disorder isgreater than what would be observed with one treatment delivered in theabsence of the other. The effect of the two treatments can be partiallyadditive, wholly additive, or greater than additive. The delivery can besuch that an effect of the first treatment delivered is still detectablewhen the second is delivered.

In some embodiments, the “at least one additional therapeutic agent”includes a TFP-expressing cell. Also provided are T-cells that expressmultiple TFPs, which bind to the same or different target antigens, orsame or different epitopes on the same target antigen. Also provided arepopulations of T-cells in which a first subset of T-cells express afirst TFP and a second subset of T-cells express a second TFP.

A TFP-expressing cell described herein and the at least one additionaltherapeutic agent can be administered simultaneously, in the same or inseparate compositions, or sequentially. For sequential administration,the TFP-expressing cell described herein can be administered first, andthe additional agent can be administered second, or the order ofadministration can be reversed.

In further aspects, a TFP-expressing cell described herein may be usedin a treatment regimen in combination with surgery, chemotherapy,radiation, immunosuppressive agents, such as cyclosporin, azathioprine,methotrexate, mycophenolate, and FK506, antibodies, or otherimmunoablative agents such as alemtuzumab, anti-CD3 antibodies or otherantibody therapies, cytoxin, fludarabine, cyclosporin, FK506, rapamycin,mycophenolic acid, steroids, FR901228, cytokines, and irradiation. ATFP-expressing cell described herein may also be used in combinationwith a peptide vaccine, such as that described in Izumoto et al. 2008 JNeurosurg 108:963-971. In a further aspect, a TFP-expressing celldescribed herein may also be used in combination with a promoter ofmyeloid cell differentiation (e.g., all-trans retinoic acid), aninhibitor of myeloid-derived suppressor cell (MDSC) expansion (e.g.,inhibitors of c-kit receptor or a VEGF inhibitor), an inhibition of MDSCfunction (e.g., COX2 inhibitors or phosphodiesterase-5 inhibitors), ortherapeutic elimination of MDSCs (e.g., with a chemotherapeutic regimensuch as treatment with doxorubicin and cyclophosphamide). Othertherapeutic agents that may prevent the expansion of MDSCs includeamino-biphosphonate, biphosphanate, sildenafil and tadalafil,nitroaspirin, vitamin D3, and gemcitabine. (See, e.g., Gabrilovich andNagaraj, Nat. Rev. Immunol, (2009) v9(3): 162-174).

In one embodiment, the subject can be administered an agent whichreduces or ameliorates a side effect associated with the administrationof a TFP-expressing cell. Side effects associated with theadministration of a TFP-expressing cell include, but are not limited tocytokine release syndrome (CRS), and hemophagocytic lymphohistiocytosis(HLH), also termed Macrophage Activation Syndrome (MAS). Symptoms of CRSinclude high fevers, nausea, transient hypotension, hypoxia, and thelike. Accordingly, the methods described herein can compriseadministering a TFP-expressing cell described herein to a subject andfurther administering an agent to manage elevated levels of a solublefactor resulting from treatment with a TFP-expressing cell. In oneembodiment, the soluble factor elevated in the subject is one or more ofIFN-γ, TNFα, IL-2, IL-6 and IL8. Therefore, an agent administered totreat this side effect can be an agent that neutralizes one or more ofthese soluble factors. Such agents include, but are not limited to asteroid, an inhibitor of TNFα, and an inhibitor of IL-6. An example of aTNFα inhibitor is entanercept. An example of an IL-6 inhibitor istocilizumab (toc).

In one embodiment, the subject can be administered an agent whichenhances the activity of a TFP-expressing cell. For example, in oneembodiment, the agent can be an agent which inhibits an inhibitorymolecule. Inhibitory molecules, e.g., Programmed Death 1 (PD1), can, insome embodiments, decrease the ability of a TFP-expressing cell to mountan immune effector response. Examples of inhibitory molecules includePD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 andTGFR beta. Inhibition of an inhibitory molecule, e.g., by inhibition atthe DNA, RNA or protein level, can optimize a TFP-expressing cellperformance. In embodiments, an inhibitory nucleic acid, e.g., aninhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA, can beused to inhibit expression of an inhibitory molecule in theTFP-expressing cell. In an embodiment the inhibitor is a shRNA. In anembodiment, the inhibitory molecule is inhibited within a TFP-expressingcell. In these embodiments, a dsRNA molecule that inhibits expression ofthe inhibitory molecule is linked to the nucleic acid that encodes acomponent, e.g., all of the components, of the TFP. In one embodiment,the inhibitor of an inhibitory signal can be, e.g., an antibody orantibody fragment that binds to an inhibitory molecule. For example, theagent can be an antibody or antibody fragment that binds to PD1, PD-L1,PD-L2 or CTLA4 (e.g., ipilimumab (also referred to as MDX-010 andMDX-101, and marketed as Yervoy™; Bristol-Myers Squibb; tremelimumab(IgG2 monoclonal antibody available from Pfizer, formerly known asticilimumab, CP-675,206)). In an embodiment, the agent is an antibody orantibody fragment that binds to TIM3. In an embodiment, the agent is anantibody or antibody fragment that binds to LAG3.

In some embodiments, the T cells may be altered (e.g., by gene transfer)in vivo via a lentivirus, e.g., a lentivirus specifically targeting aCD4+ or CD8+ T cell. (See, e.g., Zhou et al., J. Immunol. (2015)195:2493-2501).

In some embodiments, the agent which enhances the activity of aTFP-expressing cell can be, e.g., a fusion protein comprising a firstdomain and a second domain, wherein the first domain is an inhibitorymolecule, or fragment thereof, and the second domain is a polypeptidethat is associated with a positive signal, e.g., a polypeptidecomprising an intracellular signaling domain as described herein. Insome embodiments, the polypeptide that is associated with a positivesignal can include a costimulatory domain of CD28, CD27, ICOS, e.g., anintracellular signaling domain of CD28, CD27 and/or ICOS, and/or aprimary signaling domain, e.g., of CD3 zeta, e.g., described herein. Inone embodiment, the fusion protein is expressed by the same cell thatexpressed the TFP. In another embodiment, the fusion protein isexpressed by a cell, e.g., a T-cell that does not express ananti-mesothelin TFP.

In some embodiments, the human or humanized antibody domain comprisingan antigen binding domain that is an anti-mesothelin binding domainencoded by the nucliec acid, or an antibody comprising theanti-mesothelin binding domain, or a cell expressing the anti-mesothelinbinding domain encoded by the nucliec acid has an affinity value of atmost about 200 nM, 100 nM, 75 nM, a 50 nM, 25 nM, 20 nM, 15 nM, 14 nM,13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03nM, 0.02 nM, or 0.01 nM; and/or at least about 100 nM, 75 nM, a 50 nM,25 nM, 20 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM,0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM, or 0.01 nM; and or about 200 nM,100 nM, 75 nM, a 50 nM, 25 nM, 20 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM,10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM,0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM, or 0.01nM.

Pharmaceutical Compositions

Pharmaceutical compositions of the present invention may comprise aTFP-expressing cell, e.g., a plurality of TFP-expressing cells, asdescribed herein, in combination with one or more pharmaceutically orphysiologically acceptable carriers, diluents or excipients. Suchcompositions may comprise buffers such as neutral buffered saline,phosphate buffered saline and the like; carbohydrates such as glucose,mannose, sucrose or dextrans, mannitol; proteins; polypeptides or aminoacids such as glycine; antioxidants; chelating agents such as EDTA orglutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.Compositions of the present invention are in one aspect formulated forintravenous administration.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented). Thequantity and frequency of administration will be determined by suchfactors as the condition of the patient, and the type and severity ofthe patient's disease, although appropriate dosages may be determined byclinical trials.

In one embodiment, the pharmaceutical composition is substantially freeof, e.g., there are no detectable levels of a contaminant, e.g.,selected from the group consisting of endotoxin, mycoplasma, replicationcompetent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residualanti-CD3/anti-CD28 coated beads, mouse antibodies, pooled human serum,bovine serum albumin, bovine serum, culture media components, vectorpackaging cell or plasmid components, a bacterium and a fungus. In oneembodiment, the bacterium is at least one selected from the groupconsisting of Alcaligenes faecalis, Candida albicans, Escherichia coli,Haemophilus influenza, Neisseria meningitides, Pseudomonas aeruginosa,Staphylococcus aureus, Streptococcus pneumonia, and Streptococcuspyogenes group A.

When “an immunologically effective amount,” “an anti-tumor effectiveamount,” “a tumor-inhibiting effective amount,” or “therapeutic amount”is indicated, the precise amount of the compositions of the presentinvention to be administered can be determined by a physician withconsideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject). It can generally be stated that a pharmaceutical compositioncomprising the T-cells described herein may be administered at a dosageof 10⁴ to 10⁹ cells/kg body weight, in some instances 10⁵ to 10⁶cells/kg body weight, including all integer values within those ranges.T-cell compositions may also be administered multiple times at thesedosages. The cells can be administered by using infusion techniques thatare commonly known in immunotherapy (see, e.g., Rosenberg et al., NewEng. J. of Med. 319:1676, 1988).

In certain aspects, it may be desired to administer activated T-cells toa subject and then subsequently redraw blood (or have an apheresisperformed), activate T-cells therefrom according to the presentinvention, and reinfuse the patient with these activated and expandedT-cells. This process can be carried out multiple times every few weeks.In certain aspects, T-cells can be activated from blood draws of from 10cc to 400 cc. In certain aspects, T-cells are activated from blood drawsof 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc.

The administration of the subject compositions may be carried out in anyconvenient manner, including by aerosol inhalation, injection,ingestion, transfusion, implantation or transplantation. Thecompositions described herein may be administered to a patient transarterially, subcutaneously, intradermally, intratumorally, intranodally,intramedullary, intramuscularly, by intravenous (i.v.) injection, orintraperitoneally. In one aspect, the T-cell compositions of the presentinvention are administered to a patient by intradermal or subcutaneousinjection. In one aspect, the T-cell compositions of the presentinvention are administered by i.v. injection. The compositions ofT-cells may be injected directly into a tumor, lymph node, or site ofinfection.

In a particular exemplary aspect, subjects may undergo leukapheresis,wherein leukocytes are collected, enriched, or depleted ex vivo toselect and/or isolate the cells of interest, e.g., T-cells. These T-cellisolates may be expanded by methods known in the art and treated suchthat one or more TFP constructs of the invention may be introduced,thereby creating a TFP-expressing T-cell of the invention. Subjects inneed thereof may subsequently undergo standard treatment with high dosechemotherapy followed by peripheral blood stem cell transplantation. Incertain aspects, following or concurrent with the transplant, subjectsreceive an infusion of the expanded TFP T-cells of the presentinvention. In an additional aspect, expanded cells are administeredbefore or following surgery.

The dosage of the above treatments to be administered to a patient willvary with the precise nature of the condition being treated and therecipient of the treatment. The scaling of dosages for humanadministration can be performed according to art-accepted practices. Thedose for alemtuzumab, for example, will generally be in the range 1 toabout 100 mg for an adult patient, usually administered daily for aperiod between 1 and 30 days. The preferred daily dose is 1 to 10 mg perday although in some instances larger doses of up to 40 mg per day maybe used (described in U.S. Pat. No. 6,120,766).

In one embodiment, the TFP is introduced into T-cells, e.g., using invitro transcription, and the subject (e.g., human) receives an initialadministration of TFP T-cells of the invention, and one or moresubsequent administrations of the TFP T-cells of the invention, whereinthe one or more subsequent administrations are administered less than 15days, e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after theprevious administration. In one embodiment, more than one administrationof the TFP T-cells of the invention are administered to the subject(e.g., human) per week, e.g., 2, 3, or 4 administrations of the TFPT-cells of the invention are administered per week. In one embodiment,the subject (e.g., human subject) receives more than one administrationof the TFP T-cells per week (e.g., 2, 3 or 4 administrations per week)(also referred to herein as a cycle), followed by a week of no TFPT-cells administrations, and then one or more additional administrationof the TFP T-cells (e.g., more than one administration of the TFPT-cells per week) is administered to the subject. In another embodiment,the subject (e.g., human subject) receives more than one cycle of TFPT-cells, and the time between each cycle is less than 10, 9, 8, 7, 6, 5,4, or 3 days. In one embodiment, the TFP T-cells are administered everyother day for 3 administrations per week. In one embodiment, the TFPT-cells of the invention are administered for at least two, three, four,five, six, seven, eight or more weeks.

In one aspect, mesothelin TFP T-cells are generated using lentiviralviral vectors, such as lentivirus. TFP-T-cells generated that way willhave stable TFP expression.

In one aspect, TFP T-cells transiently express TFP vectors for 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15 days after transduction. Transientexpression of TFPs can be effected by RNA TFP vector delivery. In oneaspect, the TFP RNA is transduced into the T-cell by electroporation.

A potential issue that can arise in patients being treated usingtransiently expressing TFP T-cells (particularly with murine scFvbearing TFP T-cells) is anaphylaxis after multiple treatments.

Without being bound by this theory, it is believed that such ananaphylactic response might be caused by a patient developing humoralanti-TFP response, i.e., anti-TFP antibodies having an anti-IgE isotype.It is thought that a patient's antibody producing cells undergo a classswitch from IgG isotype (that does not cause anaphylaxis) to IgE isotypewhen there is a ten to fourteen-day break in exposure to antigen.

If a patient is at high risk of generating an anti-TFP antibody responseduring the course of transient TFP therapy (such as those generated byRNA transductions), TFP T-cell infusion breaks should not last more thanten to fourteen days.

EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein. Without further description,it is believed that one of ordinary skill in the art can, using thepreceding description and the following illustrative examples, make andutilize the compounds of the present invention and practice the claimedmethods. The following working examples specifically point out variousaspects of the present invention, and are not to be construed aslimiting in any way the remainder of the disclosure.

Example 1: TFP Constructs

Anti-mesothelin TFP constructs are engineered by cloning ananti-mesothelin scFv DNA fragment linked to a CD3 or TCR DNA fragment byeither a DNA sequence encoding a short linker (SL): AAAGGGGSGGGGSGGGGSLE(SEQ ID NO:2) or a long linker (LL): AAAIEVMYPPPYLGGGGSGGGGSGGGGSLE (SEQID NO:3) into p510 vector ((System Biosciences (SBI)) at XbaI and EcoR1sites.

The anti-mesothelin TFP constructs generated arep510_antimesothelin_LL_TCRa (anti-mesothelin scFv-long linker-human fulllength T-cell receptor α chain), p510_antimesothelin_LL_TCR αC(anti-mesothelin scFv-long linker-human T-cell receptor α constantdomain chain), p510_antimesothelin_LL_TCRβ (anti-mesothelin scFv-longlinker-human full length T-cell receptor β chain),p510_antimesothelin_LL_TCRβC (anti-mesothelin scFv-long linker-humanT-cell receptor3 constant domain chain), p510_antimesothelin_LL_CD3γ(anti-mesothelin scFv-long linker-human CD3γ chain),p510_antimesothelin_LL_CD36 (anti-mesothelin scFv-long linker-human CD36chain), p510_antimesothelin_LL_CD3ε (anti-mesothelin scFv-longlinker-human CD3ε chain), p510_antimesothelin_SL_TCRβ (anti-mesothelinscFv-short linker-human full length T-cell receptor3 chain),p510_antimesothelin_SL_CD3γ (anti-mesothelin scFv-short linker-humanCD3γ chain), p510_antimesothelin_SL_CD3δ (anti-mesothelin scFv-shortlinker-human CD36 chain), p510_antimesothelin_SL_CD3ε (anti-mesothelinscFv-short linker-human CD3ε chain).

The anti-mesothelin CAR construct, p510_antimesothelin_28 (is generatedby cloning synthesized DNA encoding anti-mesothelin, partial CD28extracellular domain, CD28 transmembrane domain, CD28 intracellulardomain and CD3 zeta into p510 vector at XbaI and EcoR1 sites.

Example 2: Antibody Sequences

Generation of Antibody Sequences

The human mesothelin polypeptide canonical sequence is UniProt AccessionNo. Q13421 (or Q13421-1). Provided are antibody polypeptides that arecapable of specifically binding to the human mesothelin polypeptide, andfragments or domains thereof. Anti-mesothelin antibodies can begenerated using diverse technologies (see, e.g., (Nicholson et al,1997). Where murine anti-mesothelin antibodies are used as a startingmaterial, humanization of murine anti-mesothelin antibodies is desiredfor the clinical setting, where the mouse-specific residues may induce ahuman-anti-mouse antigen (HAMA) response in subjects who receive T-cellreceptor (TCR) fusion protein (TFP) treatment, i.e., treatment withT-cells transduced with the TFP.mesothelin construct. Humanization isaccomplished by grafting CDR regions from murine anti-mesothelinantibody onto appropriate human germline acceptor frameworks, optionallyincluding other modifications to CDR and/or framework regions. Asprovided herein, antibody and antibody fragment residue numberingfollows Kabat (Kabat E. A. et al, 1991; Chothia et al, 1987).

Generation of scFvs

Human or humanized anti-mesothelin IgGs are used to generate scFvsequences for TFP constructs. DNA sequences coding for human orhumanized V_(L) and V_(H) domains are obtained, and the codons for theconstructs are, optionally, optimized for expression in cells from Homosapiens. The order in which the V_(L) and V_(H) domains appear in thescFv is varied (i.e., V_(L)—V_(H), or V_(H)—V_(L) Orientation), andthree copies of the “G₄S” or “G₄S” subunit (G₄S)₃ connect the variabledomains to create the scFv domain. Anti-mesothelin scFv plasmidconstructs can have optional Flag, His or other affinity tags, and areelectroporated into HEK293 or other suitable human or mammalian celllines and purified. Validation assays include binding analysis by FACS,kinetic analysis using Proteon, and staining of mesothelin-expressingcells.

Exemplary anti-mesothelin V_(L) and V_(H) domains, CDRs, and thenucleotide sequences encoding them, can be those described in U.S. Pat.Nos. 9,272,002; 8,206,710; 9,023,351; 7,081,518; 8,911,732; 9,115,197and 9,416,190; and U.S. Patent Publication No. 20090047211. Otherexemplary anti-mesothelin V_(L) and V_(H) domains, CDRs, and thenucleotide sequences encoding them, respectively, can be those of thefollowing monoclonal antibodies: rat anti-mesothelin antibody 420411,rat anti-mesothelin antibody 420404, mouse anti-mesothelin antibodyMN-1, mouse anti-mesothelin antibody MB-G10, mouse anti-mesothelinantibody ABIN233753, rabbit anti-mesothelin antibody FQS3796(3), rabbitanti-mesothelin antibody TQ85, mouse anti-mesothelin antibody TA307799,rat anti-mesothelin antibody 295D, rat anti-mesothelin antibody B35,mouse anti-mesothelin antibody 5G157, mouse anti-mesothelin antibody129588, rabbit anti-mesothelin antibody 11C187, mouse anti-mesothelinantibody 5B2, rabbit anti-mesothelin antibody SP74, rabbitanti-mesothelin antibody D4X7M, mouse anti-mesothelin antibody C-2,mouse anti-mesothelin antibody C-3, mouse anti-mesothelin antibody G-1,mouse anti-mesothelin antibody G-4, mouse anti-mesothelin antibody K1,mouse anti-mesothelin antibody B-3, mouse anti-mesothelin antibody200-301-A87, mouse anti-mesothelin antibody 200-301-A88, rabbitanti-mesothelin antibody EPR2685(2), rabbit anti-mesothelin antibodyEPR4509, or rabbit anti-mesothelin antibody PPI-2e(IHC).

In some embodiments, single-domain (V_(HH)) binders are used such asthose set forth in SEQ ID NOS 58, 59, and 55 (SD1, SD4, and SD6,respectively).

Source of TCR Subunits

Subunits of the human T Cell Receptor (TCR) complex all contain anextracellular domain, a transmembrane domain, and an intracellulardomain. A human TCR complex contains the CD3-epsilon polypeptide, theCD3-gamma polypeptide, the CD3-delta polypeptide, the CD3-zetapolypeptide, the TCR alpha chain polypeptide and the TCR beta chainpolypeptide. The human CD3-epsilon polypeptide canonical sequence isUniprot Accession No. P07766. The human CD3-gamma polypeptide canonicalsequence is Uniprot Accession No. P09693. The human CD3-deltapolypeptide canonical sequence is Uniprot Accession No. P043234. Thehuman CD3-zeta polypeptide canonical sequence is Uniprot Accession No.P20963. The human TCR alpha chain canonical sequence is UniprotAccession No. Q6ISU1. The human TCR beta chain C region canonicalsequence is Uniprot Accession No. P01850, a human TCR beta chain Vregion sequence is P04435.

The human CD3-epsilon polypeptide canonical sequence is:

(SEQ ID NO: 4) MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSI SGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDED HLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCE NCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAK PVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYS  GLNQRRI.

The human CD3-gamma polypeptide canonical sequence is:

(SEQ ID NO: 5) MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLY QPLKDREDDQYSHLQGNQLRRN.

The human CD3-delta polypeptide canonical sequence is:

( SEQ ID NO: 6) MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQY SHLGGNWARNKS.

The human CD3-zeta polypeptide canonical sequence is:

(SEQ ID NO: 7) MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

The human TCR alpha chain canonical sequence is:

(SEQ ID NO: 8) MAGTWLLLLLALGCPALPTGVGGTPFPSLAPPIMLLVDGKQQ MVVVCLVLDVAPPGLDSPIWFSAGNGSALDAFTYGPSPATDG TWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHLSGEASTARTCPQEPLRGTPGGALWLGVLRLLLFKLLLFDLLLT CSCLCDPAGPLPSPATTTRLRALGSHRLHPATETGGREATSS PRPQPRDRRWGDTPPGRKPGSPVWGEGSYLSSYPTCPAQAWCSRSALRAPSSSLGAFFAGDLPPPLQAGAA.

The human TCR alpha chain C region canonical sequence is:

(SEQ ID NO: 9) PNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRI LLLKVAGFNLLMTLRLWSS.

The human TCR alpha chain V region CTL-L17 canonical sequence is:

(SEQ ID NO: 10) MAMLLGASVLILWLQPDWVNSQQKNDDQQVKQNSPSLSVQEGRISILNCDYTNSMFDYFLWYKKYPAEGPTFLISISSIKDKNEDGRFTVFLNKSAKHLSLHIVPSQPGDSAVYFCAAKGAGT ASKLTFGTGTRLQVTL.

The human TCR beta chain C region canonical sequence is:

(SEQ ID NO: 11) EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVS ALVLMAMVKRKDF.

The human TCR beta chain V_region CTL-L17 canonical sequence is:

(SEQ ID NO: 12) MGTSLLCWMALCLLGADHADTGVSQNPRHNITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSLAGLNQPQHFGD GTRLSIL.

The human TCR beta chain V region YT35 canonical sequence is:

(SEQ ID NO: 13) MDSWTFCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGHNSLFWYRQTMMRGLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSFSTCSANYG YTFGSGTRLTVV.Generation of TFPs from TCR Domains and scFvs

The mesothelin scFvs are recombinantly linked to CD3-epsilon or otherTCR subunits (see 1C) using a linker sequence, such as G₄S, (G₄S)₂(G₄S)₃ or (G₄S)₄. Various linkers and scFv configurations are utilized.TCR alpha and TCR beta chains were used for generation of TFPs either asfull length polypeptides or only their constant domains. Any variablesequence of TCR alpha and TCR beta chains is allowed for making TFPs.

TFP Expression Vectors

Expression vectors are provided that include: a promoter(Cytomegalovirus (CMV) enhancer-promoter), a signal sequence to enablesecretion, a polyadenylation signal and transcription terminator (BovineGrowth Hormone (BGH) gene), an element allowing episomal replication andreplication in prokaryotes (e.g., SV40 origin and ColE1 or others knownin the art) and elements to allow selection (ampicillin resistance geneand zeocin marker).

Preferably, the TFP-encoding nucleic acid construct is cloned into alentiviral expression vector and expression validated based on thequantity and quality of the effector T-cell response ofTFP.mesothelin-transduced T-cells (“mesothelin.TFP” or “mesothelin.TFPT-cells” or “TFP.mesothelin” or “TFP.mesothelin T-cells”) in response tomesothelin+ target cells. Effector T-cell responses include, but are notlimited to, cellular expansion, proliferation, doubling, cytokineproduction and target cell lysis or cytolytic activity (i.e.,degranulation).

The TFP.mesothelin lentiviral transfer vectors are used to produce thegenomic material packaged into the VSV-G pseudotyped lentiviralparticles. Lentiviral transfer vector DNA is mixed with the threepackaging components of VSV-G, gag/pol and rev in combination withLipofectamine® reagent to transfect them together into HEK-293(embryonic kidney, ATCC® CRL-1573™) cells. After 24 and 48 hours, themedia is collected, filtered and concentrated by ultracentrifugation.The resulting viral preparation is stored at −80° C. The number oftransducing units is determined by titration on Sup-T1 (T-celllymphoblastic lymphoma, ATCC® CRL-1942™) cells. RedirectedTFP.mesothelin T-cells are produced by activating fresh naive T-cellswith, e.g., anti-CD3 anti-CD28 beads for 24 hrs and then adding theappropriate number of transducing units to obtain the desired percentageof transduced T-cells.

These modified T-cells are allowed to expand until they become restedand come down in size at which point they are cryopreserved for lateranalysis. The cell numbers and sizes are measured using a CoulterMultisizer™ III. Before cryopreserving, the percentage of cellstransduced (expressing TFP.mesothelin on the cell surface) and therelative fluorescence intensity of that expression are determined byflow cytometric analysis. From the histogram plots, the relativeexpression levels of the TFPs are examined by comparing percentagetransduced with their relative fluorescent intensity.

In some embodiments multiple TFPs are introduced by T-cell transductionwith multiple viral vectors.

Evaluating Cytolytic Activity, Proliferation Capabilities and CytokineSecretion of Humanized TFP Redirected T Cells

The functional abilities of TFP.mesothelin T-cells to producecell-surface expressed TFPs, and to kill target tumor cells, proliferateand secrete cytokines are determined using assays known in the art.Human peripheral blood mononuclear cells (PBMCs, e.g., blood from anormal apheresed donor whose naive T-cells are obtained by negativeselection for T-cells, CD4+ and CD8+ lymphocytes) are treated with humaninterleukin-2 (IL-2) then activated with anti-CD3x anti-CD28 beads,e.g., in 10% RPMI at 37° C., 5% CO₂ prior to transduction with theTFP-encoding lentiviral vectors. Flow cytometry assays are used toconfirm cell surface presence of a TFP, such as by an anti-FLAG antibodyor an anti-munne variable domain antibody. Cytokine (e.g., IFN-γ)production is measured using ELISA or other assays.

Example 3: Human TFP T-Cell Efficacy in a Human ALL Mouse Model

Primary human ALL cells can be grown in immune compromised mice (e.g.,NSG or NOD) without having to culture them in vitro. Likewise, culturedhuman ALL cell lines can induce leukemia in such mice. ALL-bearing micecan be used to test the efficacy of human TFP.mesothelin T-cells, forinstance, in the model HALLX5447. The readout in this model is thesurvival of mice after intravenous (i.v.) infusion of ALL cells in theabsence and presence of i.v. administered human TFP.mesothelin T-cells.

Example 4: Demonstration of Multiplexed TFP Polypeptides, and Use ofMultiplexed Humanized TFP Redirected T-Cells

The TFP polypeptides provided herein are capable of functionallyassociating with endogenous TCR subunit polypeptides to form functionalTCR complexes. Here, multiple TFPs in lentiviral vectors are used totransduce T-cells in order to create a functional, multiplexedrecombinant TCR complex. For example, provided is a T-cell containing i)a first TFP having an extracellular domain, a transmembrane domain, andan intracellular domain from the CD3-delta polypeptide and amesothelin-specific scFv antibody fragment, and ii) a second TFP havingan extracellular domain, a transmembrane domain, and an intracellulardomain from the CD3-gamma polypeptide and a mesothelin-specific antibodyfragment. The first TFP and second TFP are capable of interacting witheach other and with endogenous TCR subunit polypeptides, thereby forminga functional TCR complex.

The use of these multiplexed humanized TFP.mesothelin T-cells can bedemonstrated in liquid and solid tumors as provided in Examples 2 and 3above.

Example 5: Preparation of T-cells Transduced with TFPs

Lentiviral Production

Lentivirus encoding the appropriate constructs are prepared as follows.5×10⁶ HEK-293FT-cells are seeded into a 100 mm dish and allowed to reach70-90% confluency overnight. 2.5 g of the indicated DNA plasmids and 20μL Lentivirus Packaging Mix (ALSTEM, cat #VP100) are diluted in 0.5 mLDMEM or Opti-MEM® I Medium without serum and mixed gently. In a separatetube, 30 μL of NanoFect® transfection reagent (ALSTEM, cat #NF100) isdiluted in 0.5 mL DMEM or Opti-MEM® I Medium without serum and mixedgently. The NanoFect/DMEM and DNA/DMEM solutions are then mixed togetherand votrexed for 10-15 seconds prior to incubation of theDMEM-plasmid-NanoFect mixture at room temperature for 15 minutes. Thecomplete transfection complex from the previous step is added dropwiseto the plate of cells and rocked to disperse the transfection complexevenly in the plate. The plate is then incubated overnight at 37° C. ina humidified 5% CO₂ incubator. The following day, the supernatant isreplaced with 10 mL fresh media and supplemented with 20 μL ofViralBoost (500×, ALSTEM, cat #VB100). The plates are then incubated at37° C. for an additional 24 hours. The lentivirus containing supernatantis then collected into a 50 mL sterile, capped conical centrifuge tubeand put on ice. After centrifugation at 3000 rpm for 15 minutes at 4°C., the cleared supernatant is filtered with a low-protein binding 0.45m sterile filter and virus is subsequently isolated byultracentrifugation at 25,000 rpm (Beckmann, L8-70M) for 1.5 hours, at4° C. The pellet is removed and re-suspended in DMEM media andlentivirus concentrations/titers are established by quantitative RT-PCR,using the Lenti-X qRT-PCR Titration kit (Clontech; catalog number631235). Any residual plasmid DNA is removed by treatment with DNaseI.The virus stock preparation is either used for infection immediately oraliquoted and stored at −80° C. for future use.

PBMC Isolation

Peripheral blood mononuclear cells (PBMCs) are prepared from eitherwhole blood or buffy coat. Whole blood is collected in 10 mL Heparinvacutainers and either processed immediately or stored overnight at 4°C. Approximately 10 mL of whole anti-coagulated blood is mixed withsterile phosphate buffered saline (PBS) buffer for a total volume of 20mL in a 50 mL conical centrifuge tube (PBS, pH 7.4, without Ca²⁺/Mg²⁺).20 mL of this blood/PBS mixture is then gently overlaid onto the surfaceof 15 mL of Ficoll-Paque® PLUS (GE Healthcare, 17-1440-03) prior tocentrifugation at 400 g for 30-40 min at room temperature with no brakeapplication.

Buffy coat is purchased from Research Blood Components (Boston, Mass.).LeucoSep® tubes (Greiner bio-one) are prepared by adding 15 mLFicoll-Paque® (GE Health Care) and centrifuged at 1000 g for 1 minute.Buffy coat is diluted 1:3 in PBS (pH 7.4, without Ca²⁺ or Mg²⁺). Thediluted buffy coat is transferred to Leucosep tube and centrifuged at1000 g for 15 minutes with no brake application. The layer of cellscontaining PBMCs, seen at the diluted plasma/ficoll interface, isremoved carefully to minimize contamination by ficoll. Residual ficoll,platelets, and plasma proteins are then removed by washing the PBMCsthree times with 40 mL of PBS by centrifugation at 200 g for 10 minutesat room temperature. The cells are then counted with a hemocytometer.The washed PBMC are washed once with CAR-T media (AIM V-AlbuMAX® (BSA)(Life Technologies), with 5% AB serum and 1.25 μg/mL amphotericin B(Gemini Bioproducts, Woodland, Calif.), 100 U/mL penicillin, and 100μg/mL streptomycin). Alternatively, the washed PBMC's are transferred toinsulated vials and frozen at −80° C. for 24 hours before storing inliquid nitrogen for later use.

T-Cell Activation

PBMCs prepared from either whole blood or buffy coat are stimulated withanti-human CD28 and CD3 antibody-conjugated magnetic beads for 24 hoursprior to viral transduction. Freshly isolated PBMC are washed once inCAR-T media (AIM V-AbuMAX (BSA) (Life Technologies), with 5% AB serumand 1.25 μg/mL amphotericin B (Gemini Bioproducts), 100 U/mL penicillin,and 100 μg/mL streptomycin) without huIL-2, before being re-suspended ata final concentration of 1×10⁶ cells/mL in CAR-T medium with 300 IU/mLhuman IL-2 (from a 1000× stock; Invitrogen). If the PBMCs had previouslybeen frozen they are thawed and re-suspended at 1×10⁷ cells/mL in 9 mLof pre-warmed (37° C.) cDMEM media (Life Technologies), in the presenceof 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin, at aconcentration of 1×10⁶ cells/mL prior to washing once in CAR-T medium,re-suspension at 1×10⁶ cells/mL in CAR-T medium, and addition of IL-2 asdescribed above.

Prior to activation, anti-human CD28 and CD3 antibody-conjugatedmagnetic beads (available from, e.g., Invitrogen, Life Technologies) arewashed three times with 1 mL of sterile 1×PBS (pH 7.4), using a magneticrack to isolate beads from the solution, before re-suspension in CAR-Tmedium, with 300 IU/mL human IL-2, to a final concentration of 4×10⁷beads/mL. PBMC and beads are then mixed at a 1:1 bead-to-cell ratio, bytransferring 25 μL (1×10⁶ beads) of beads to 1 mL of PBMC. The desirednumber of aliquots are then dispensed to single wells of a 12-welllow-attachment or non-treated cell culture plate, and incubated at 37°C., with 5% CO₂, for 24 hours before viral transduction.

T-Cell Transduction/Transfection and Expansion

Following activation of PBMC, cells are incubated for 48 hours at 37°C., 5% CO₂. Lentivirus is thawed on ice and 5×10⁶ lentivirus, along with2 μL of TransPlus™ (Alstem) per mL of media (a final dilution of 1:500)is added to each well of 1×10⁶ cells. Cells are incubated for anadditional 24 hours before repeating addition of virus. Alternatively,lentivirus is thawed on ice and the respective virus is added at 5 or 50MOI in presence of 5 μg/mL polybrene (Sigma). Cells are spinoculated at100 g for 100 minutes at room temperature. Cells are then grown in thecontinued presence of 300 IU/mL of human IL-2 for a period of 6-14 days(total incubation time is dependent on the final number of CAR-T-cellsrequired). Cell concentrations are analyzed every 2-3 days, with mediabeing added at that time to maintain the cell suspension at 1×10⁶cells/mL.

In some instances, activated PBMCs are electroporated with in vitrotranscribed (IVT) mRNA. In one embodiment, human PBMCs are stimulatedwith Dynabeads® (ThermoFisher) at 1-to-1 ratio for 3 days in thepresence of 300 IU/ml recombinant human IL-2 (R&D Systems) (otherstimulatory reagents such as TransAct T Cell Reagent from MilyeniPharmaceuticals may be used). The beads are removed beforeelectroporation. The cells are washed and re-suspended in OPTI-MEMmedium (ThermoFisher) at the concentration of 2.5×10⁷ cells/mL. 200 μLof the cell suspension (5×10⁶ cells) are transferred to the 2 mm gapElectroporation Cuvettes Plus™ (Harvard Apparatus BTX) and prechilled onice. 10 μg of IVT TFP mRNA is added to the cell suspension. ThemRNA/cell mixture is then electroporated at 200 V for 20 millisecondsusing ECM830 Electro Square Wave Porator (Harvard Apparatus BTX).Immediately after the electroporation, the cells are transferred tofresh cell culture medium (AIM V AlbuMAX (BSA) serum free medium+5%human AB serum+300 IU/ml IL-2) and incubated at 37° C.

Verfication of TFP Expression by Cell Staining

Following lentiviral transduction or mRNA electroporation, expression ofanti-mesothelin TFPs is confirmed by flow cytometry, using an anti-mouseFab antibody to detect the murine anti-mesothelin scFv. T-cells arewashed three times in 3 mL staining buffer (PBS, 4% BSA) andre-suspended in PBS at 1×106 cells per well. For dead cell exclusion,cells are incubated with LIVE/DEAD® Fixable Aqua Dead Cell Stain(Invitrogen) for 30 minutes on ice. Cells are washed twice with PBS andre-suspended in 50 μL staining buffer. To block Fc receptors, 1 μL of1:100 diluted normal goat lgG (BD Bioscience) is added to each tube andincubated in ice for 10 minutes. 1.0 mL FACS buffer is added to eachtube, mixed well, and cells are pelleted by centrifugation at 300 g for5 min. Surface expression of scFv TFPs is detected by Zenon®R-Phycoerythrin-labeled human MSLN IgG1 Fc or human IgG1 isotypecontrol. 1 μg antibodies are added to the respective samples andincubated for 30 minutes on ice. Cells are then washed twice, andstained for surface markers using Anti-CD3 APC (clone, UCHT1),anti-CD4-Pacific blue (Clone RPA-T4), nti-CD8 APCCy7(Clone SKi), from BDbioscience. Flow cytometry is performed using LSRFortessa™ X20 (BDBiosciences) and data is acquired using FACS diva software and isanalyzed with FlowJo® (Treestar, Inc. Ashland, Oreg.).

Exemplary results are shown in FIG. 5A, which shows the surfaceexpression analysis of activated PBMC cells stained for CD8 (anti-CD8APCCy7, y-axes) and mesothelin (“MSLN”) (Zenon® R-Phycoerythrin-labeledhMSLN IgG, x-axes). Shown from left to right are cells that were eithernon-transduced or transduced with anti-MSLN-CD3ε, anti-MSLN-CD28ζ, andanti-MSLN-41BB(constructs. The proportion of CD8+, MSLN+ cells is shownin the top right corner of each panel.

FIG. 5B shows similar results from activated PBMC cells, stained forMSLN and GFP, that were transduced with TFP constructs comprisingin-house single domain (“SD”) mesothelin binders. The top row shows(from left to right) non-transduced cells, and cells transduced with apositive control anti-MSLN-CD3ε TFP (“SS1”). Rows 2-4 show the anti-MSLNbinders SD1, SD4, and SD6, respectively, in cells transduced withGFP-tagged (from left to right) CD3ε TFP, CD3γTFP, TCRβ TFP, and CD28ζCAR constructs. The proportion of GFP+, MSLN+ cells is shown in the topright corner of each panel.

Example 6: Cytotoxicity Assay by Flow Cytometry

Target cells that are either positive or negative for mesothelin arelabelled with the fluorescent dye, carboxyfluorescein diacetatesuccinimidyl ester (CFSE). These target cells are mixed with effectorT-cells that are either un-transduced, transduced with control CAR-Tconstructs, or transduced with TFPs. After the indicated incubationperiod, the percentage of dead to live CFSE-labeled target cells andnegative control target cells is determined for each effector/targetcell culture by flow cytometry. The percent survival of target cells ineach T-cell-positive target cell culture is calculated relative to wellscontaining target cells alone.

The cytotoxic activity of effector T-cells is measured by comparing thenumber of surviving target cells in target cells without or witheffector T-cells, following co-incubation of effector and target cells,using flow cytometry. In experiments with mesothelin TFPs orCAR-T-cells, the target cells are mesothelin-positive cells, while cellsused as a negative control are mesothelin-negative cells.

Target cells are washed once, and re-suspended in PBS at 1×10⁶ cells/mL.The fluorescent dye carboxyfluorescein diacetate succinimidyl ester(CFSE) (ThermoFisher) is added to the cell suspension at a concentrationof 0.03 M and the cells are incubated for 20 minutes at roomtemperature. The labeling reaction is stopped by adding to the cellsuspension complete cell culture medium (RPMI-1640+10% HI-FBS) at thevolume 5 times of the reaction volume, and the cells are incubated foran additional two minutes at room temperature. The cells are pelleted bycentrifugation and re-suspended in cytotoxicity medium (Phenol red-freeRPMI1640 (Invitrogen) plus 5% AB serum (Gemini Bioproducts) at 2×10⁵cells/mL. Fifty microliters of CFSE labelled-target cell suspension(equivalent to 10,000 cells) are added to each well of the 96-wellU-bottom plate (Corning).

Effector T-cells transduced with anti-mesothelin TFP constructs,together with non-transduced T-cells as negative controls, are washedand suspended at 2×10⁶ cells/mL, or 1×10⁶ cells/mL in cytotoxicitymedium. 50 μL of effector T-cell suspensions (equivalent to 100,000 or50,000 cells) are added to the plated target cells to reach theeffector-to-target ratio of 10-to-1 or 5-to-1, respectively, in a totalvolume of 100 μL. The cultures are then mixed, spun down, and incubatedfor four hours at 37° C. and 5% CO₂. Immediately following thisincubation, 7AAD (7-aminoactinomycin D) (BioLegend) is added to thecultured cells as recommended by the manufacturer, and flow cytometry isperformed with a BD LSRFortessa™ X-20 (BD Biosciences). Analysis of flowcytometric data is performed using FlowJo® software (TreeStar, Inc.).

The percentage of survival for target cells is calculated by dividingthe number of live target cells (CFSE+7-AAD-) in a sample with effectorT-cells and target cells, by the number of live (CFSE+7-AAD-) cells inthe sample with target cells alone. The cytotoxicity for effector cellsis calculated as the percentage of killing for targetcells=100%-percentage of survival for the cells.

T-cells transduced with an anti-MSLN 28ζ CAR construct may demonstratecytotoxicity against mesothelin-expressing cells when compared toT-cells that are either non-transduced or are transduced with anon-mesothelin-specific CAR control. However, T-cells transduced withanti-mesothelin-CD3ε may induce more efficient cytotoxicity against thetargets than the anti-mesothelin CAR control. Anti-mesothelin-CD3γ TFPsmay also mediate robust cytotoxicity that is greater than that observedwith anti-mesothelin-CAR at effector:target ratios between 5 and 10:1.Some cytotoxicity may be observed with anti-mesothelin-TCRα andanti-mesothelin-TCRβ TFPs. Similar results may be obtained withanti-mesothelin TFPs constructed with an alternative hinge region. Onceagain, cytotoxicity against mesothelin-expressing target cells may begreater with anti-mesothelin-CD3ε or anti-mesothelin-CD3γ TFP-transducedT-cells than with anti-mesothelin-CAR-transduced T-cells.

T-cells electroporated with mRNA encoding TFPs specific for mesothelinmay also demonstrate robust cytotoxicity against mesothelin-expressingcells. While no significant killing of the mesothelin-negative cells maybe seen with either control or anti-mesothelin TFP constructs,mesothelin-specific killing of mesothelin-expressing cells may beobserved by T-cells transduced with either anti-mesothelin-CD3ε SL, oranti-mesothelin-CD3γ SL TFPs.

Example 8: Determining Cytotoxicity by Real Time Cytotoxicity Assay

Anti-mesothelin TFPs may also demonstrate superior cytotoxicity overanti-mesothelin CARs in the real-time cytotoxicity assay (RTCA) format.The RTCA assay measures the electrical impedance of an adherent targetcell monolayer, in each well of a specialized 96-well plate, in realtime and presents the final readout as a value called the cell index.Changes in cell index indicate disruption of the target cell monolayeras a result of killing of target cells by co-incubated T-cell effectors.Thus the cytotoxicity of the effector T-cells can be evaluated as thechange in cell index of wells with both target cells and effectorT-cells compared to that of wells with target cells alone.

Adherent target cells are cultured in DMEM, 10% FBS, 1%Antibiotic-Antimycotic (Life Technologies). To prepare the RTCA, 50 μLof, e.g., DMEM medium is added into the appropriate wells of an E-plate(ACEA Biosciences, Inc, Catalog #: JL-10-156010-1A). The plate is thenplaced into a RTCA MP instrument (ACEA Biosciences, Inc.) and theappropriate plate layout and assay schedule entered into the RTCA 2.0software as described in the manufacturers manual. Baseline measurementis performed every 15 minutes for 100 measurements. 1×10⁴ target cellsin a 100 μL volume are then added to each assay well and the cells areallowed to settle for 15 minutes. The plate is returned to the readerand readings are resumed.

The next day, effector T-cells are washed and re-suspended incytotoxicity media (Phenol red-free RPMI1640 (Invitrogen) plus 5% ABserum (Gemini Bioproducts; 100-318)). The plate is then removed from theinstrument and the effector T-cells, suspended in cytotoxicity medium(Phenol red-free RPMI1640+5% AB serum), are added to each well at100,000 cells or 50,000 cells to reach the effector-to-target ratio of10-to-1 or 5-to-1, respectively. The plate is then placed back to theinstrument. The measurement is carried out for every 2 minutes for 100measurements, and then every 15 minutes for 1,000 measurements.

In the RTCA assay, killing of mesothelin-transduced cells may beobserved by T-cells transduced with anti-mesothelin-28ζ CAR-transducedT-cells, as demonstrated by a time-dependent decrease in the cell indexfollowing addition of the effector cells relative to cells alone orcells co-incubated with T-cells transduced with a control CAR construct.However, target cell killing by anti-mesothelin-CD3ε TFP-expressingT-cells may be deeper and more rapid than that observed with theanti-mesothelin CAR. For example, within 4 hours of addition of T-cellstransduced with anti-mesothelin-CD3ε TFP, killing of themesothelin-expressing target cells may be essentially complete. Littleor no killing may be observed with T-cells transduced with a number ofTFP constructs comprising other CD3 and TCR constructs. Similar resultsmay be obtained with anti-mesothelin TFPs constructed with analternative hinge region. Cytotoxicity against mesothelin-transducedtarget cells may be greater with anti-mesothelin-CD3ε oranti-mesothelin-CD3γ TFP-transduced T-cells than withanti-mesothelin-CAR-transduced T-cells.

The cytotoxic activity of TFP-transduced T-cells may be dose-dependentwith respect to the amount of virus (MOI) used for transduction.Increased killing of mesothelin-positive cells may be observed withincreasing MOI of anti-mesothelin-CD3ε TFP lentivirus, furtherreinforcing the relationship between TFP transduction and cytotoxicactivity.

Exemplary results of the RTCA assay are shown in FIGS. 6A-C. Ananti-MSLN TFP construct was engineered by cloning an anti-MSLN scFv DNAfragment linked to a CD3ε DNA fragment by a DNA sequence coding thelinker: GGGGSGGGGSGGGGSLE (SEQ ID NO:1) into a p510 vector (from SBI) atXbaI and EcoRI sites. The anti-MSLN TFP construct generated wasp510_antiMSLN_SS1_CD3a (anti-MSLN SS1 scFv-linker-human CD3ε chain).

Full length mesothelin (NM_005823) was PCR amplified frompCMV6_XL4_Mesothelin (Origene) and cloned into XbaI and EcoRIrestriction digested p527a (pCDH-EF-MCS-T2A-Puro) (SBI) via GibsonRecombination reaction.

Target cells for the RTCA were mesothelin-positive HeLa cells (cervicaladenocarcinoma, ATCC® CCL-2™) and mesothelin-negative PC-3 cells(prostate adenocarcinoma, ATCC® CRL-1435™) were used as negativecontrols. Adherent target cells were cultured in DMEM with 10% FBS and1% Antibiotic-Antimycotic (Life Technologies).

The normalized cell index, indicative of cytotoxicity, was determined.Activated PBMCs were untreated (trace #1), non-transduced (trace #2), ortransduced with empty vector (trace #3), an anti-MSLN TFP(“Anti-MSLN-CD3ε TRuC”, trace #4), an anti-MSLN CAR with the CD28((trace#5) or 41BB((trace #6) signaling doman (“Anti-MSLN-28ζ CAR” and“Anti-MSLN-41BBζ CAR,” respectively).

As shown in FIG. 6A, the target MSLN-positive HeLa cells wereefficiently killed by the anti-MSLN TFP-transduced T cells, compared tothe negative controls. In contrast, the MSLN-negative PC-3 cells werenot efficiently killed by any of the constructs (FIG. 6B).

A similar experiment was performed using in-house TFP constructs withsingle-domain anti-mesothelin binders. FIG. 6C shows killing ofMSLN-positive cells in a high target density cell line(HeLa-(MSLN^(high))) using T cells from two different human donors (topand bottom). Shown are the cell killing traces for TFP T cells with theanti-MSLN binders SD1 (left), SD4 (middle), and SD6 (right). ActivatedPBMCs were non-transduced (trace #1), ortransduced with CD3ε TFP (trace#2), CD3γ TFP (trace #3), TCRβ TFP (trace #4), or CD28ζ CAR (trace #5).The normalized cell index, indicative of cytotoxicity, was determined ina real time cell analyzer (RTCA) assay. As shown in the Figure, all theT cells, except the non-transduced, were able to kill cancer cells.

Example 7: Luciferase-Based Cytotoxicity Assay in Cells with High or LowTarget Density

The luciferase-based cytotoxicity assay (“Luc-Cyto” assay) assesses thecytotoxicity of TFP T and CAR T cells by indirectly measuring theluciferase enzymatic activity in the residual live target cells afterco-culture. The high target density cells used in Luc-Cyto assay wereHeLa-MSLN^(high) cells and the low target density cells used were PC3cells expressing low levels of mesothelin (PC3-MSLN^(low)), each stablytransduced to express firefly luciferase. The DNA encoding fireflyluciferase was synthesized by GeneArt® (Thermo Fisher®) and insertedinto the multiple cloning site of single-promoter lentiviral vectorpCDH527A-1 (System Bioscience).

The lentivirus carrying the firefly luciferase was packaged as describedabove. The HeLa-MSLN^(high) or PC3-MSLN^(low) cells were then transducedwith the firefly luciferase construct carrying lentivirus for 24 hoursand then selected with puromycin (5 μg/mL). The generation ofHeLa-luc-MSLN^(high)- and PC3-luc-MSLN^(low)-luciferase cells wasconfirmed by measuring the luciferase enzymatic activity in the cellswith Bright-Glo™ Luciferase Assay System (Promega). Separate populationsfrom two different human donors were transduced with an empty expressionvector (“NT”), or the following TFPs or CARs: anti-MSLN (positivecontrol, “SS1”, affinity 11 nM), anti-MSLN-SD1 (affinity 25 nM),anti-MSLN-SD4 (affinity 6 nM), or anti-MSLN SD6 (affinity 0.59 nM), eachin the format of CD3ε TFP, CD3γ TFP, TCR TFP, and CD28 ζ CAR. The twopopulations of transduced T cells were incubated with HeLa-MSLN^(high)(FIGS. 7A-C) or PC3-MSLN^(low) (FIGS. 8A-D).

The target cells were plated at 5000 cells per well in 96-well plate.The TFP T, the CAR T, or control cells were added to the target cells ateffector-to-target ratios or 1:1 (black bars) or 1:5 (gray bars). Themixture of cells was then cultured for 24 hours at 37° C. with 5% CO₂before the luciferase enzymatic activity in the live target cells wasmeasured by the Bright-Glo® Luciferase Assay System. The cells were spuninto a pellet and resuspended in medium containing the luciferasesubstrate. Luciferase is released by cell lysis, thus, higher luciferaseactivity corresponds to a greater percentage of cell death.

Results using cells expressing high levels of MSLN are shown in FIGS.7A-C. Shown are the % of cells killed in samples with no T cells(“target only”), empty vector transduced (“NT”), anti-MSLN (positivecontrol, “SS1”), or anti-mesothelin TFP T cells with in-houseanti-mesothelin binders SD1 (FIG. 7A), SD4 (FIG. 7B), and SD6 (FIG. 7C),each in each in the format of CD3ε TFP, CD3γ TFP, TCRβ TFP, and CD28ζCAR. In each graph, black bars represent a 1:1 ratio of T cells totarget cells, and gray bars represent a 1:5 ratio of T cells to targetcells. As can be seen in the Figures, all of the TFP-T cells, CAR-Tcells, and positive control SS1 were efficient at killing the MSLN

FIGS. 8A-D are a series of graphs showing the activity of anti-MSLN CART cells and TFP T cells against a target cell line expressing low levelsof mesothelin (PC3-MSLN^(low)). Shown are the % of cells killed insamples with no T cells (“target only”), empty vector transduced (“NT”),anti-MSLN (positive control, “SS1”), or anti-mesothelin constructs SD1,SD4, and SD6 in the TFP formats CD3ε (FIG. 8A), CD3γ (FIG. 8B), TCRβ(FIG. 8C), and CD28ζ CAR (FIG. 8D). In each graph, black bars representa 1:1 ratio of T cells to target cells, and gray bars represent a 1:5ratio of T cells to target cells. Similar results were seen for a secondT cell donor.

As shown in the FIG., a 1:1 ratio of T cells to target cells resulted inthe highest level of killing of target cells, as was expected. Inaddition, all TFP T and CAR T cells showed similar activity in cellsexpressing high levels of MSLN.

Example 8: Measurement of Activation of T Cells by FACS

Activation of the T-cells expressing anti-MSLN CAR and TFP Constructswas performed using MSLN+ and MSLN-K562 cells, and is shown in FIGS.9A-D. As described above, Activated PBMCs were transduced with 50 MOILVs for two consecutive days and expanded. Day 8 post transduction,co-cultures of PBMCs were set up with target cells (K562 cellsoverexpressing MSLN) at E:T, 1:1 ratio (0.2×10⁶ each cell type) incytotoxicity medium (Phenol red-free RPMI1640 (Invitrogen) plus 5% ABserum (Gemini Bioproducts; 100-318). K562 cells overexpressing BCMA wereused as negative controls. 24 hours after the beginning of co-culturing,cells were harvested, washed with PBS three times and stained withLive/Dead Aqua for 30 min on ice. To block Fc receptors, human Fc block(BD) was added and incubated for 10 minutes at room temperature. Cellswere subsequently stained with anti-CD3 APC (clone, UCHT1), anti-CD8APCcy7(Clone SKi), anti-CD69-Alexa Fluor® 700 (clone FN50) from BDBiosciences and anti-CD25-PE (Clone BC96, eBioscience). Cells werewashed twice and analyzed by BD LSRII-Fortessa. Data were analyzed asabove using FlowJo® analysis software (Tree star, Inc.).

As shown in FIG. 9A, from left to right, T cells were eithernon-transduced, transduced with empty vector, transduced withanti-MSLN-CD3ε TFP, anti-MSLN-28ζ CAR, or anti-MSLN-41BBζ CAR. Cellsco-cultured with MSLN− cells are shown in the top row, and thoseco-cultured with MSLN+ target cells are shown in the bottom row. Thecells were then stained with antibodies specific for the surfaceactivation markers CD69 and CD25. The numbers of cells stained withanti-CD69 correspond to the x-axes and those stained with anti-CD25correspond to the y-axes. As shown, T-cells expressing anti-mesothelinCAR and TFP constructs were activated by culturing with MSLN+ cells, asdemonstrated by elevated levels of CD69 and CD25 expression, relative toco-culturing with MSLN− cells (FIG. 9B). The percentage of CD25+ cellsfor each construct in MSLN− (white bars) and MSLN+(black bars) cells isshown.

A similar experiment was done using K562 MSLN− cells (FIG. 9C, circles)and K562-MSLN+ cells (FIG. 9C, squares) in either non-transduced T cellsor T cells transduced with anti-MSLN positive control binders(“510-SS1-CD3ε). Data represent the sum of CD25+, CD69+, and CD25+/CD69+cells. In FIG. 9D, data are shown for the in-house anti-MSLN binders SD1(squares), SD4 (circles), and SD6 (triangles) in K562 MSLN− target cells(left panel) and K562 MSLN+ cells (right panel) combined with donor Tcells having TFP formats CD3ε, CD3γ, TCRβ, and CD28ζ CAR. Similarresults were seen using cells from a second T cell donor.

Activation of T-cells may be similarly assessed by analysis of granzymeB production. T-cells are cultured and expanded as described above, andintracellular staining for granzyme B is done according to themanufacturer's kit instructions (Gemini Bioproducts; 100-318). cellswere harvested, washed with PBS three times and blocked with human Fcblock for 10 min. Cells were stained for surface antigens with anti-CD3APC (clone, UCHT1), and anti-CD8 APCcy7(Clone SKi) for 30 min at 4° C.Cells were then fixed with Fixation/Permeabilization solution (BDCytofix/Cytoperm Fixation/Permealbilzation kit cat #554714) for 20 minat 4 C, flowed by washing with BD Perm/Wash buffer. Cells weresubsequently stained with anti-Granzyme B Alexafluor700 (Clone GB11),washed with BD Perm/Wash buffer twice and resuspended in FACS buffer.Data was acquired on BD LSRII-Fortessa and analyzed using FlowJo® (Treestar Inc.).

As shown in FIG. 10A, from left to right, T cells were eithernon-transduced, transduced with empty vector, transduced withAnti-MSLN-CD3ε TFP, anti-MSLN-28ζ CAR, or anti-MSLN-41BB(CAR. Cellsco-cultured with MSLN− cells are shown in the top row, and thoseco-cultured with MSLN+ target cells are shown in the bottom row. Thenumbers of cells stained with anti-GrB correspond to the x-axes andthose stained with anti-CD8 correspond to the y-axes. As shown, T-cellsexpressing anti-mesothelin CAR and TFP constructs were activated byculturing with MSLN+ cells, but not the MSLN− cells. These results areshown again in FIG. 10B, wherein the percentage of GrB+ cells for eachconstruct in mesothelin negative (“MSLN−”, white bars) and mesothelinpositive (“MSLN+, black bars) cells is shown. These data demonstrate theability of MSLN-expressing cells to specifically activate T-cells.

Example 9: IL-2 and IFN-γ Secretion by ELISA

Another measure of effector T-cell activation and proliferationassociated with the recognition of cells bearing cognate antigen is theproduction of effector cytokines such as interleukin-2 (IL-2) andinterferon-gamma (IFN-γ).

ELISA assays for human IL-2 (catalog #EH2IL2, Thermo Scientific) andIFN-γ catalog #KHC4012, Invitrogen) are performed as described in theproduct inserts. In one example, 50 μL of reconstituted standards orsamples in duplicate are added to each well of a 96-well plate followedby 50 μL of Biotinylated Antibody Reagent. Samples are mixed by gentlytapping the plate several times. 50 μL of Standard Diluent is then addedto all wells that did not contain standards or samples and the plate iscarefully sealed with an adhesive plate cover prior to incubation for 3hours at room temperature (20-25° C.). The plate cover is then removed,plate contents are emptied, and each well is filled with Wash Buffer.This wash procedure is repeated a total of 3 times and the plate isblotted onto paper towels or other absorbent material. 100 μL ofprepared Streptavidin-HRP Solution is added to each well and a new platecover is attached prior to incubation for 30 minutes at roomtemperature. The plate cover is again removed, the plate contents arediscarded, and 100 μL of TMB Substrate Solution is added into each well.The reaction is allowed to develop at room temperature in the dark for30 minutes, after which 100 μL of Stop Solution is added to each well.Evaluate the plate. Absorbance is measured on an ELISA plate reader setat 450 nm and 550 nm within 30 minutes of stopping the reaction. 550 nmvalues are subtracted from 450 nm values and IL-2 amounts in unknownsamples are calculated relative to values obtained from an IL-2 standardcurve.

Alternatively, 2-Plex assays are performed using the Human CytokineMagnetic Buffer Reagent Kit (Invitrogen, LHB0001M) with the Human IL-2Magnetic Bead Kit (Invitrogen, LHC0021M) and the Human IFN-γ MagneticBead Kit (Invitrogen, LHC4031M). Briefly, 25 μL of Human IL-2 and IFN-γantibody beads are added to each well of a 96-well plate and washedusing the following guidelines: two washes of 200 μL 1× wash solution,placing the plate in contact with a Magnetic 96-well plate Separator(Invitrogen, A14179), letting the beads settle for 1 minute anddecanting the liquid. Then, 50 μL of Incubation Buffer is added to eachwell of the plate with 100 μL of reconstituted standards in duplicatesor 50 μL of samples (supernatants from cytotoxicity assays) and 50 μL ofAssay Diluent, in triplicate, for a total volume of 150 μL. Samples aremixed in the dark at 600 rpm with an orbital shaker with a 3 mm orbitalradius for 2 hours at room temperature. The plate is washed followingthe same washing guidelines and 100 μL of human IL-2 and IFN-γbiotinylated detector antibody is added to each well. Samples are mixedin the dark at 600 rpm with an orbital shaker with a 3 mm orbital radiusfor 1 hour at room temperature. The plate is washed following the samewashing guidelines and 100 μL of Streptavidin-R-Phycoerythrin is addedto each well. Samples are mixed in the dark at 600 rpm with an orbitalshaker with a 3 mm orbital radius for 30 minutes at room temperature.The plate is washed 3 times using the same washing guidelines and afterdecanting the liquid the samples are re-suspended in 150 μL of 1× washsolution. The samples are mixed at 600 rpm with an orbital shaker with a3 mm orbital radius for 3 minutes and stored over night at 4° C.Afterwards, the plate is washed following the same washing guidelinesand the samples are re-suspended in 150 μL of 1× wash solution.

The plate is read using the MAGPIX System (Luminex) and xPONENTsoftware. Analysis of the data is performed using MILLIPLEX Analystsoftware, which provides the standard curve and cytokine concentrations.

Relative to non-transduced or control CAR-transduced T-cells, T-cellstransduced with anti-mesothelin TFPs may produce higher levels of bothIL-2 and IFN-γ when co-cultured with either cells that endogenouslyexpress mesothelin or mesothelin-transduced cells. In contrast,co-culture with mesothelin negative cells or non-transduced cells, mayresult in little or no cytokine release from TFP-transduced T-cells.Consistent with the previous cytotoxicity data, anti-mesothelin TFPsconstructed with an alternative hinge region may generate similarresults upon co-culture with mesothelin-bearing target cells.

In agreement with the previous cytotoxicity data, anti-mesothelin-CD3εand anti-mesothelin-CD3γ may produce the highest IL-2 and IFN-γ levelsof the TFP constructs. However, cytokine production by T-cellstransduced with anti-mesothelin-CD3ε and anti-mesothelin-CD3γ TFPs maybe comparable to that of T-cells expressing anti-mesothelin-28ζ CAR,despite the TFPs demonstrating much higher levels of target cellkilling. The possibility that TFPs may more efficiently kill targetcells than CARs, but release comparable or lower levels ofpro-inflammatory cytokines, represents a potential advantage for TFPsrelative to CARs since elevated levels of these cytokines have beenassociated with dose-limiting toxicities for adoptive CAR-T therapies.

Exemplary results are shown in FIGS. 11A-B. As described above,activated PBMCs were transduced with 50 MOI lentiviruses for twoconsecutive days and expanded. Day 8 post transduction, co-cultures ofPBMCs were set up with target cells (K562 cells overexpressing MSLN) atE:T, 1:1 ratio (0.2×10⁶ each cell type) in cytotoxicity medium (Phenolred-free RPM11640 (Invitrogen) plus 5% AB serum (Gemini Bioproducts;100-318). K562 cells overexpressing BCMA were used as negative controls.After 24 hours cells were analyzed for IFN-γ (FIG. 11A) and IL-2 (FIG.11B) expression by ELISA as described above. In each FIG., from left toright, T cells were either non-transduced, transduced with empty vector,transduced with Anti-MSLN-CD3ε TFP, anti-MSLN-28ζ CAR, oranti-MSLN-41BB(CAR. Cells co-cultured with MSLN− cells are representedby white bars, and those co-cultured with MSLN+ target cells arerepresented by black bars. As can be seen in the FIG., T-cellsexpressing anti-mesothelin CAR and TFP constructs were activated, asevidenced by both IFN-γ and IL-2 production, by co-culturing with MSLN+cells, but not the MSLN− cells, further demonstrating the ability ofMSLN-expressing cells to specifically activate T-cells.

Example 10: CD107a Exposure by Flow Cytometry

An additional assay for T-cell activation is surface expression ofCD107a, a lysosomal associated membrane protein (also known as LAMP-1)that is located in the membrane of cytoplasmic cytolytic granules inresting cells. Degranulation of effector T-cells, a prerequisite forcytolytic activity, results in mobilization of CD107a to the cellsurface following activation-induced granule exocytosis. Thus, CD107aexposure provides an additional measure of T-cell activation, inaddition to cytokine production, that correlates closely withcytotoxicity.

Target and effector cells are separately washed and re-suspended incytotoxicity medium (RPMI+5% human AB serum+1% antibiotic antimycotic).The assay is performed by combining 2×10⁵ effectors cells with 2×10⁵target cells in a 100 μL final volume in U-bottom 96-well plates(Corning), in the presence of 0.5 μL/well of PE/Cy7-labelled anti-humanCD107a (LAMP-1) antibody (Clone-H4A3, BD Biosciences). The cultures arethen incubated for an hour at 37° C., 5% CO₂. Immediately following thisincubation, 10 μL of a 1:10 dilution of the secretion inhibitor monensin(1000× solution, BD GolgiStop™) is carefully added to each well withoutdisturbing the cells. The plates are then incubated for a further 2.5hours at 37° C., 5% CO₂. Following this incubation, the cells arestained with APC anti-human CD3 antibody (Clone-UCHT1, BD Biosciences),PerCP/Cy5.5 anti-human CD8 antibody (Clone-SKi, BD Biosciences) andPacific Blue anti-human CD4 antibody (Clone-RPA-T4, BD Biosciences) andthen incubated for 30 minutes at 37° C., 5% CO₂. The cells are thenwashed 2× with FACS buffer (and resuspended in 100 μL FACS buffer and100 ul IC fix buffer prior to analysis.

Exposure of CD107a on the surface of T-cells is detected by flowcytometry. Flow cytometry is performed with a LSRFortessa™ X20 (BDBiosciences) and analysis of flow cytometric data is performed usingFlowJo software (Treestar, Inc. Ashland, Oreg.). The percentage of CD8+effector cells, within the CD3 gate, that are CD107+ve is determined foreach effector/target cell culture.

Consistent with the previous cytotoxicity and cytokine data, co-cultureof mesothelin-expressing target cells with effector T-cells transducedwith anti-mesothelin-28ζ CAR may induce an increase in surface CD107aexpression relative to effectors incubated with mesothelin negativetarget cells. In comparison, under the same conditions,anti-mesothelin-CD3ε LL or anti-mesothelin-CD3γ LL TFP-expressingeffectors may exhibit a 5- to 7-fold induction of CD107a expression.Anti-mesothelin TFPs constructed with an alternative hinge region maygenerate similar results upon co-culture with mesothelin-bearing targetcells.

Example 11: In Vivo Mouse Efficacy Studies

To assess the ability of effector T-cells transduced withanti-mesothelin TFPs to achieve anti-tumor responses in vivo, effectorT-cells transduced with either anti-mesothelin-28ζ CAR,anti-mesothelin-CD3ε LL TFP or anti-mesothelin-CD3γ LL TFP areadoptively transferred into NOD/SCID/IL-2Rγ−/− (NSG-JAX) mice that hadpreviously been inoculated with mesothelin+ human cancer cell lines.

Female NOD/SCID/IL-2Rγ−/− (NSG-JAX) mice, at least 6 weeks of age priorto the start of the study, are obtained from The Jackson Laboratory(stock number 005557) and acclimated for 3 days before experimental use.Human mesothelin-expressing cell lines for inoculation are maintained inlog-phase culture prior to harvesting and counting with trypan blue todetermine a viable cell count. On the day of tumor challenge, the cellsare centrifuged at 300 g for 5 minutes and re-suspended in pre-warmedsterile PBS at either 0.5-1×10⁶ cells/100 μL. T-cells for adoptivetransfer, either non-transduced or transduced with anti-mesothelin-28CAR, anti-mesothelin-CD3ε LL TFP or anti-CD3γ LL TFP constructs areprepared. On day 0 of the study, 10 animals per experimental group arechallenged intravenously with 0.5-1×10⁶ mesothelin-expressing cells. 3days later, 5×10⁶ of effector T-cell populations are intravenouslytransferred to each animal in 100 μL of sterile PBS. Detailed clinicalobservations on the animals are recorded daily until euthanasia. Bodyweight measurements are made on all animals weekly until death oreuthanasia. All animals are euthanized 35 days after adoptive transferof test and control articles. Any animals appearing moribund during thestudy are euthanized at the discretion of the study director inconsultation with a veterinarian.

Relative to non-transduced T-cells, adoptive transfer of T-celltransduced with either anti-mesothelin-28ζ CAR, anti-mesothelin-CD3ε LLTFP or anti-mesothelin-CD3γ LL TFP may prolong survivalmesolthelin-expressing cell line tumor-bearing mice, and may indicatethat both anti-mesothelin CAR- and TFP-transduced T-cells are capable ofmediating target cell killing with corresponding increased survival inthese mouse models. Collectively, these data may indicate that TFPsrepresent an alternative platform for engineering chimeric receptorsthat demonstrate superior antigen-specific killing to first generationCARs both in vitro and in vivo.

Example 12: Human TFP T-Cell Treatment in an In Vivo Solid TumorXenograft Mouse Model

The efficacy of treatment with human TFP.mesothelin T-cells can also betested in immune compromised mouse models bearing subcutaneous solidtumors derived from human mesothelin-expressing ALL, CLL, NHL, or MSTOhuman cell lines. Tumor shrinkage in response to treatment with humanTFP.mesothelin T-cells can be either assessed by caliper measurement oftumor size or by following the intensity of a green fluorescence protein(GFP) signal emitted by GFP-expressing tumor cells.

Primary human solid tumor cells can be grown in immune compromised micewithout having to culture them in vitro. Exemplary solid cancer cellsinclude solid tumor cell lines, such as provided in The Cancer GenomeAtlas (TCGA) and/or the Broad Cancer Cell Line Encyclopedia (CCLE, seeBarretina et al., Nature 483:603 (2012)). Exemplary solid cancer cellsinclude primary tumor cells isolated from mesothelioma, renal cellcarcinoma, stomach cancer, breast cancer, lung cancer, ovarian cancer,prostate cancer, colon cancer, cervical cancer, brain cancer, livercancer, pancreatic cancer, kidney, endometrial, or stomach cancer. Insome embodiments, the cancer to be treated is selected from the groupconsisting of mesotheliomas, papillary serous ovarian adenocarcinomas,clear cell ovarian carcinomas, mixed Mullerian ovarian carcinomas,endometroid mucinous ovarian carcinomas, pancreatic adenocarcinomas,ductal pancreatic adenocarcinomas, uterine serous carcinomas, lungadenocarcinomas, extrahepatic bile duct carcinomas, gastricadenocarcinomas, esophageal adenocarcinomas, colorectal adenocarcinomasand breast adenocarcinomas. These mice can be used to test the efficacyof TFP.mesothelin T-cells in the human tumor xenograft models (see,e.g., Morton et al., Nat. Procol. 2:247 (2007)). Following an implant orinjection of 1×10⁶-1×10⁷ primary cells (collagenase-treated bulk tumorsuspensions in EC matrix material) or tumor fragments (primary tumorfragments in EC matrix material) subcutaneously, tumors are allowed togrow to 200-500 mm³ prior to initiation of treatment.

One such experiment was performed to test the efficacy of MSLN-specificsingle domain antibody (sdAb) activity in vivo in a mesotheliomaxenograft mouse model as described above. Luciferase-labeledMSTO-211H-FL-MSLN-Luc) were inoculated at 1×10⁶ cells per mouse,subcutaneously, as a 1:1 ratio with Matrigel®. Tumor volume wasmonitored by caliper measurement twice weekly. Fourteen days after tumorinjection, when tumor volume was approximately 300 mm³, 1×107 T cellswere injected intravenously into each animal. T cells used includedthose transduced with a CD3ε-SD1 TFP, a CD3γ-SD1 TFP, a CD3ε-SD4 TFP, aCD3γ-SD4 TFP, a CD28(SD1 CAR, and a CD28(SD1 CAR. A group of mice withno T cell injection was used as a negative control.

Results are shown in FIG. 12A. Mice injected with CD3ε-SD1 TFP andCD3γ-SD1 TFP T cells showed the greatest and fastest reduction in tumorvolume, although mice injected with any but the no T cell control showedreductions in tumor volume after the injection of the T cells.

The persistent efficacy of SD1 ε- and γ-TFP T cells was tested in vivoby rechallenging the surviving mice in the mesothelioma xenograft mousemodel.

The mice were inoculated with 1×10⁶ tumor cells (MSTO 211H FL MSLN Luc)per mouse, subcutaneously, with Matrigel® (to-1 ratio). One group ofmice were injected with Raji cells as a negative control, and one groupof mice was injected with MSTO cells alone, again as a negative control.Tumor volume was monitored by caliper measurement twice a week. Fourteendays after tumor injection (when tumor volume reached approximately 300mm³), 1×10⁷ MSTO (MSLN+) or Raji (MSLN-, as a negative control) wereinjected intravenously into each animal. Results are shown in FIG. 12B.Each line in the figure represents single animal. As shown in the FIG.,mice that had previously been treated with anti-MSLN TFP T cells wereable to again reduce tumor volume or eradicate the tumor, indicatingthat either the originally injected T cells persisted in the mice, orthat the mice had developed an anti-MSLN memory response. In contrast,mice re-challenged with Raji (MSLN-) cells were not able to control thegrowth of the Raji tumors, thus illustrating the specificity of the TFPT-cell response.

Example 13. In Vivo Efficacy of Patients' Derived MSLN ε-TFP T Cells inMSLN Tumor Xenograft Mouse Model

SD1 ε-TFP T cells from ovarian cancer patients were used to test the invitro and in vivo anti-tumor efficacy of SD1 ε-TFP T cells againstmesothelin expressing tumor cells (MSTO-MSLN-Luc).

Lentivirus was prepared as described above.

Preparation of CD4⁺ and CD8⁺ T Cells from Whole Blood of Ovarian CancerPatients

CD4⁺ and CD8+ T cells were purified from whole blood of ovarian cancerpatients as follows (a schematic overview is shown in FIG. 13A). 40-50mL of heparinized whole blood of ovarian cancer patients was collectedand shipped overnight by Conversant Bio (Huntsville, Ala.). The bloodwas diluted with an equal volume of PBS and 35 mL of diluted whole bloodwas carefully layered over 15 mL of Ficoll-Paque® (GE healthcare, cat #:17-5442-02) in a 50 mL conical tube. It was then centrifuged at 800×gfor 20 min at RT in a swinging bucket rotor without brake. The upperlayer was aspirated, leaving the mononuclear cell layer (lymphocytes,monocytes, and thrombocytes) undisturbed at the interphase. Themononuclear cell layer was transferred to a new 50 mL conical tube, add30 mL of PBS and centrifuge at 300×g for 10 min at RT. 1-2 mL of ACKlysis buffer was added (ThermoFisher, cat #: A1049201) to the pellets,mixed thoroughly, and incubated at RT for 2 min, 20 mL of PBS was added,centrifuged at 300×g for 10 min at RT. Cell pellets were resuspended in10 mL of ice cold MACs buffer and cells were counted via a CellometerAuto 2000. CD4+ and CD8+ T cell isolation was performed using Miltenyihuman CD4/8 microbeads (cat #: 130-045-101; 130-045-201) according tomanufacturers' instructions.

TFP T cells were produced as described above, and transduction wasdetermined by FACS. Mesothelin expression was confirmed on target cells(MSLNigh cell line MSTO-211H-FL MSLN (generated in house from parentalMSTO-211H, ATCC, CRL-2081)) and MSLN-Fc expression was confirmed SD1ε-TFP T cells by flow cytometry on the same day as a luciferase assay.The single suspension of luciferase-labeled target cells (MSTO-211H-FLMSLN-Luc or the MSLN− cell line C30-Luc (A2780, Sigma)) was prepared inR10 medium. 1×10⁴ of target cells in 100 μL was added to 96-wellflat-bottom plate. TFP T cells were added in 100 μL at differenteffector-to-target ratio (E:T) as indicated.

FACS-Based Transduction Efficiency and T Cell Activation Determination

TFP T cells were thawed, debeaded (if ex vivo expanded in Dynabeads+IL-2condition), washed, and then re-suspended in T cell culture mediawithout cytokine. The desired number of T cells (in 100 μL) was added toreach effector-to-target ratio at 5-to-1, 1-to-1 and 1-to-5,respectively. Three replicates were prepared for each type of T cell attested ratio. The cells were then cultured for 24 hours at 37° C. with5% CO₂. After 24 hours' co-culture, the plate was centrifuged at 300×gfor 2 minutes to pellet down the cells. 100 μL of culture supernatantfrom each well were removed carefully for Luminex assay. 100 μL of assaybuffer from Bright-Glo™ Luciferase Assay System (Promega, #E2650) wereadded to each well. The content in each well was mixed by gentlypipetting up and down. The cell-reagent mixture was left at roomtemperature in dark for 3 minutes for complete lysis of the cells. 200μL of cell lysate from each well were transferred to Greiner-One whitewalled 96 well plate. The luminescence was measured relativeluminescence unit (RLU) by SpectraMax M5 plate reader (Moleculardevices).

The percent (%) of tumor lysis was calculated by the formula listedbelow:

${\%\mspace{14mu}{Tumor}\mspace{14mu}{Lysis}} = {100*\left\lbrack {1 - \frac{{Luminescence}\mspace{11mu}\left( {{Tumor} + {T\mspace{11mu}{cell}}} \right)}{{Luminescence}\;({Tumor})}} \right\rbrack}$Luminex® Assay

Supernatant from tumor-T cell co-culture was harvest and stored in −80°C. as described previously. Cytokine profiles were detected usingMillipore Luminex kit (HCD8MAG-15K) as according to manufacturers'instructions. The supernatant was plated without any dilution and thereading was measured using a Magpix xMAP® Technology.

Subcutaneous Mesothelioma Xenograft Mouse Model and In Vivo Assessments

Female 6-week-old NSG mice (NOD.Cg-Prkdc^(scid) Il2rg^(tm1Wjl)/SzJ, cat#: 005557, Jackson Laboratories) were used in this study. The animalswere acclimated for minimum 3 days under the same condition as thestudy. The MSTO-211H-FLMSLN-Luc cells were suspended in sterile PBS at aconcentration of 1×10⁶ cells/100 μL. The PBS cell suspension was thenmixed 1-to-1 with ice cold Matrigel® for a final injection volume of 200μL for each mouse. The resulting PBS/Matrigel® cell suspension was kepton ice until subcutaneous administration in the dorsal hind flank of themouse. Tumor growth was monitored as tumor volume with Calipermeasurement. The volume of tumor was calculated as:Tumor volume=½(length×width²)

Ten days after tumor cell injection, the animals were randomizedaccording to tumor volume (200˜300 mm³) and divided into 10 groups toreceive injection of SD1 ε-TFP T cells from different patients (numberof mice per group varies depending on the number of SD1 ε-TFP T cellsrecovered on the day of injection). The T cell injection day wasconsidered as the day 0 of the study. The T cells were prepared insterile PBS at a concentration of 5×10⁶ cells/100 μL. The cellsuspension was then injected intravenously into the mouse via tail vein.

Ex vivo expansion of SD1 ε-TFP T cells from ovarian cancer patients

MSLN-specific sdAb TFP T cells were prepared with lentivirus encodingCD3ε formats of the TFP with SD1 binders targeting MSLN. Fold expansion,determined by viable cell count on day 10, ranged from 8.58 to 28.2 fold(17.8+/−3.3) compared to day 0 in cells prepared with Dynabeads®+IL-2,and 10 to 33.6 fold (22.9+/−5.0) compared to day 0 in cells preparedwith TransAct®+IL-7/15. The transduction efficiency for the SD1 ε-TFP Tcells was determined on day 10 of expansion by surface stain for thepresence of GFP and MSLN-Fc on CD4⁺ and CD8⁺ populations. Transductionefficiency ranged from 28.6% to 52.1% (40.9+/−4.0%) in cells preparedwith Dynabeads+IL-2, and 5.7% to 46.9% (26.8+/−6.3%) in cells preparedwith TransAct+IL-7/15; no significant differences were shown in foldexpansion and transduction efficiency between Dynabeads+IL-2 andTransAct+IL-7/15 conditions. Vector copy number per cell was in linewith transduction efficiency, with around 1˜2 copy numbers per cell ineither Dynabeads+IL-2 or TransAct+IL7/15 conditions, except for patient1, which had 0.38 vector copy number per cell.

In Vitro Anti-Tumor Activity of SD1 ε-TFP T Cells from Ovarian CancerPatients

The in vitro efficacy of SD1 ε-TFP T cells from ovarian cancer patientswas tested using luciferase reporter tumor cell lysis assays. Mesothelinexpression was confirmed on MSTO-211H-FLMSLN-Luc cell lines on the dayof assay (FIG. 13B); all SD1 ε-TFP T cells showed different levels oftumor killing. Robust tumor cell lysis was observed for patients 1, 2,4, and 5 (75%97%). MSLN ε-TFP T cells when co-cultured withMSTO-211H-FLMSLN-Luc (a MSLN high expresser) at 5-to-1 effector totarget ratio, patient 3 shows ˜35% of tumor lysis at 5-to-1 effector totarget ratio, 4 out of 5 patients (patients 1, 2, 4, and 5) showed onaverage 50% of tumor lysis at 1-to-1 effector to target ratio, 2 out of5 (patients 4 and 5) showed ˜50% of tumor lysis even at 1-to-5 effectorto target ratio. All T cells showed rapid killing of the tumor cell. Notumor lysis was observed for all MSLN ε-TFP™ T cells when co-culturedwith mesothelin negative cell lines C30-Luc (FIG. 13C). The cytokineprofile of MSLN ε-TFP from five patients were analyzed using a human CD8Luminex® panel, cytolytic cytokines such as IFN-γ, GM-CSF, Granzyme-A/B,IL-2, MIP-1α/β, TNF-α, and perforin were significantly increased in MSLNε-TFP™ T cells compared to non-transduced T cells (FIGS. 13D-L).

In Vivo Efficacy of MSLN ε-TFP T Cells in MSLN-Expressing Tumor MouseXenograft Model

MSTO-211H-FLMSLN-Luc was used to establish a subcutaneous xenograftedmesothelin-expressing tumor mouse model. Tumor volume was measured twicea week. On day 10 post tumor injection, the average tumor volume reached200-300 mm³, and day 10-expanded MSLN ε-TFP T cells from one normaldonor (ND12, FIG. 14A) and patients 1-4 (FIGS. 14B-E) and were thawedand transduction efficiency was confirmed. 5×10⁶ per mouse MSLN ε-TFP Tcells or matching non-transduced T cells were i.v. injected and tumorvolumes were monitored thereafter. MSLN ε-TFP T cells from 3 out of 4patients (patients 1, 2, and 4) showed complete tumor clearance by day20 post-T cell injection. Tumor clearance was maintained until day 40.Five out of six mice received MSLN ε-TFP T cells from patient 3, whichshowed partial protection. From all four patients who received MSLNε-TFP T cells from ND12, one showed complete tumor clearance, two showedpartial tumor clearance.

ENDNOTES

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

APPENDIX A: SEQUENCE SUMMARY SEQ  ID  NO.  Name  Sequence  1 Short Linker 1  GGGGSGGGGSGGGGSLE  2  Short Linker 2 AAAGGGGSGGGGSGGGGSLE  3  Long Linker  AAAIEVMYPPPYLGGGGSGGGGSGGGGSLE  4 human CD3-ϵ MQSGTHVVRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVIL TCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGY YVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGG LLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYE  PIRKGQRDLYSGLNQRRI  5 human CD3-γ  MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDA EAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKS KPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVR QSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN  6  human CD3-δMEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGT LLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVEL DPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQ VYQPLRDRDDAQYSHLGGNWARNKS  7  human CD3-ζMKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALF LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEM GGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY  QGLSTATKDTYDALHMQALPPR 57  human TCR   MAGTVVLLLLLALGCPALPTGVGGTPFPSLAPPIMLLVDGKQQMVVVC α-chain  LVLDVAPPGLDSPIWFSAGNGSALDAFTYGPSPATDGTVVTNLAHLSLP SEELASWEPLVCHTGPGAEGHSRSTQPMHLSGEASTARTCPQEPLRGT PGGALWLGVLRLLLFKLLLFDLLLTCSCLCDPAGPLPSPATTTRLRALG SHRLHPATETGGREATSSPRPQPRDRRWGDTPPGRKPGSPVWGEGSYL SSYPTCPAQAWCSRSALRAPSSSLGAFFAGDLPPPLQAGA  9  human TCR  PNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKT  α-chain C  VLDMRSMDFKSNSAVAVVSNKSDFACANAFNNSIIPEDTFFPSPESSCDV  regionKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS  10  human TCR  MAMLLGASVLILWLQPDWVNSQQKNDDQQVKQNSPSLSVQEGRISIL  α-chain V NCDYTNSMFDYFLWYKKYPAEGPTFLISISSIKDKNEDGRFTVFLNKSA  region  CTL-L17 KHLSLHIVPSQPGDSAVYFCAAKGAGTASKLTFGTGTRLQVTL  11  human TCR  EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVN  β-chain C GKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRC  region QVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGV LSATILYEILLGKATLYAVLVSALVLMAMVKRKDF  12  human TCR  MGTSLLCWMALCLLGADHADTGVSQNPRHNITKRGQNVTFRCDPISE  β-chain V  HNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFST  region CTL-L17 LEIQRTEQGDSAMYLCASSLAGLNQPQHFGDGTRLSIL  13  human TCR  MDSWTFCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGH  β-chain V NSLFWYRQTMMRGLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTL  region YT35 KIQPSEPRDSAVYFCASSFSTCSANYGYTFGSGTRLTVV  14  MSLN DNA acgcgtgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatga  Seq. gttagcaacatgccttacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacgatcgtgccttattaggaaggcaacagacgggtctgac atggattggacgaaccactgaattgccgcattgcagagatattgtatttaagtgcctagctcgatacaataaacgggtctctctggttagaccagatctgagcctg ggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgc cttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactag agatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccg aacagggacctgaaagcgaaagggaaaccagagctctctcgacgcaggactcg gcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgc caaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggcc agggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagc tagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaagaacttag atcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagaga taaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagt aagaccaccgcacagcaagcggccactgatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaa ccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaa aagagcagtgggaataggagctttgttccttgggttcttgggagcagcaggaa gcactatgggcgcagcctcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaaca gcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcc tggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaa taaatctctggaacagattggaatcacacgacctggatggagtgggacagaga aattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaacca gcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtg gaattggtttaacataacaaattggctgtggtatataaaattattcataatgat agtaggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaa tagagttaggcagggatattcaccattatcgtttcagacccacctcccaacccc gaggggacccgacaggcccgaaggaatagaagaagaaggtggagagagagaca gagacagatccattcgattagtgaacggatctcgacggtatcggttaactttt  aaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaattca aaattttatcgatactagtattatgcccagtacatgaccttatgggactttcc tacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggt tttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttcca agtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacg ggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggta ggcgtgtacggtgggaggtttatataagcagagctcgtttagtgaaccgtcag atcgcctggagacgccatccacgctgttttgacctccatagaagattctagag ccgccaccatgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccagacattcagcaggtccagctccagcagtctgg ccctgaactcgaaaaacctggcgctagcgtgaaaatttcctgtaaagcctccg gctactcttttactggctacacaatgaattgggtgaaacagtctcacggcaaa tccctcgaatggatcggactcatcacaccctacaatggcgcctcttcctacaa ccagaaattccggggcaaggcaacactcactgtggacaaatcatcctctaccg cctacatggatctgctctccctcacatctgaggactccgctgtctacttttgt gcccgaggaggatacgacggacgaggattcgattactggggacagggaacaac tgtgaccgtgtctagtggcggcggagggagtggaggcggaggatcttctggcg ggggatccgatattgaactcacacagtctcccgctatcatgtctgcttctccc ggcgagaaagtgactatgacttgctctgcttcctcttctgtgtcctacatgca ctggtaccagcagaaatctggcacatcccctaaacggtggatctacgatactagcaaactggcatccggcgtgcctgggcgattctctggctctggctctggcaac tcttactctctcacaatctcatctgtcgaggctgaggacgatgccacatacta ctgtcagcagtggtctaaacacccactcacattcggcgctggcactaaactggaaataaaagcggccgcaggtggcggcggttctggtggcggcggttctggtggc ggcggttctctcgaggatggtaatgaagaaatgggtggtattacacagacacc atataaagtctccatctctggaaccacagtaatattgacatgccctcagtatc ctggatctgaaatactalggcaacacaatgataaaaacataggcggtgatgag gatgataaaaacataggcagtgatgaggatcacctgtcactgaaggaattttc agaattggagcaaagtggttattatgtctgctaccccagaggaagcaaaccag aagatgcgaacttttatctctacctgagggcaagagtgtgtgagaactgcatggagatggatgtgatgtcggtggccacaattgtcatagtggacatctgcatcac tgggggcttgctgctgctggtttactactggagcaagaatagaaaggccaagg ccaagcctgtgacacgaggagcgggtgctggcggcaggcaaaggggacaaaacaaggagaggccaccacctgttcccaacccagactatgagcccatccggaaagg ccagcgggacctgtattctggcctgaatcagagacgcatctgataagaattcg atccgcggccgcgaaggatctgcgatcgctccggtgcccgtcagtgggcagag  cgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaacgggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtact ggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtc gccgtgaacgttctttttcgcaacgggtttgccgccagaacacagctgaagct  tcgaggggctcgcatctctccttcacgcgcccgccgccctacctgaggccgccatccacgccggttgagtcgcgttctgccgcctcccgcctgtggtgcctcctga actgcgtccgccgtctaggtaagtttaaagctcaggtcgagaccgggcctttg tccggcgctcccttggagcctacctagactcagccggctctccacgctttgcctgaccctgcttgctcaactctacgtcmgtttcgttttctgttctgcgccgttac agatccaagctgtgaccggcgcctacgctagatgaccgagtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgtacgcaccctcgccgcc gcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccgccacat cgagcgggtcaccgagctgcaagaactcttcctcacgcgcgtcgggctcgaca tcggcaaggtgtgggtcgcggacgacggcgccgcggtggcggtctggaccacg ccggagagcgtcgaagcgggggcggtgttcgccgagatcggcccgcgcatggc cgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctcctgg cgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcggcgtctcg cccgaccaccagggcaagggtctgggcagcgccgtcgtgctccccggagtgga ggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgca acctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtg cccgaaggaccgcgcacctggtgcatgacccgcaagcccggtgcctgagtcga caatcaacctctggattacaaaatttgtgaaagattgactggtattcttaact  atgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcgtatggctttcattttctcctccttgtataaatcctggttgc tgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtca gctcctttccgggactttcgctttccccctccctattgccacggcggaactca tcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgaca attccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccct caatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttc cgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcctggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaacgaaaa taagatctgctttttgcttgtactgggtctctctggttagaccagatctgagc ctgggagctctctggctaactagggaacccactgcttaagcctcaataaagct tgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaac tagagatccctcagacccttttagtcagtgtggaaaatctctagcagtagtag  ttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttattgcagcttataatggttacaaataaagcaat agcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggctctagctatcccgcc cctaactccgcccagttccgcccattctccgccccatggctgactaatttttt ttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtag tgaggaggcttttttggaggcctagacttttgcagagacggcccaaattcgta atcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccac acaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtg agctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaa cctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtt tgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtc gttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatc cacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaa aggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgc ccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaaccc gacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgta ggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgacc gctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctt tgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacag ttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttc atccatagttgcctgactccccgtcgtgtagataactacgatacgggagggctt accatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctcc agatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcc tgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagt aagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcat cgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacg atcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttc tgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgacc gagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaac tttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggat cttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatc ttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatact cttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatt tccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaac ctataaaaataggcgtatcacgaggccctttcgtctcgcgcgtttcggtgatga cggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgca ccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcag gcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggc  ctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaag ttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgc  caagctg  15 MSLN amino  MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEAAPL acid sequence:  DGVLANPPNISSLSPRQLLGFPCAEVSGLSTERVRELAVALAQKNVKLS human  TEQLRCLAHRLSEPPEDLDALPLDLLLFLNPDAFSGPQACTRFFSRITKA  mesothelin NVDLLPRGAPERQRLLPAALACWGVRGSLLSEADVRALGGLACDLPG  sequence RFVAESAEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPSTWSV  (UniProt STMDALRGLLPVLGQPIIRSIPQGIVAAWRQRSSRDPSWRQPERTILRPR  Accession No. FRREVEKTACPSGKKAREIDESLIFYKKWELEACVDAALLATQMDRV  Q13421) NAIPFTYEQLDVLKHKLDELYPQGYPESVIQHLGYLFLKMSPEDIRKW NVTSLETLKALLEVNKGHEMSPQVATLIDRFVKGRGQLDKDTLDTLT AFYPGYLCSLSPEELSSVPPSS1WAVRPQDLDTCDPRQLDVLYPKARLA FQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDA VLPLTVAEVQKLLGPHVEGLKAEERHRPVRDVVILRQRQDDLDTLGLG LQGGIPNGYLVLDLSMQEALSGTPCLLGPGPVLTVLALLLASTLA  16  p510_anti- acgcgtgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgag  MSLN_SS1_CD ttagcaacatgccttacaaggagagaaaaagcaccgtgcatgccgattggtgga  3ϵ DNA agtaaggtggtacgatcgtgccttattaggaaggcaacagacgggtctgacatg gattggacgaaccactgaattgccgcattgcagagatattgtatttaagtgcctagctcgatacaataaacgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgag tgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccc tcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacctgaaagcgaaagggaaaccagagctctctcgacgcaggactcggcttgctga agcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaa aaaatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggaca gctacaaccatcccttcagacaggatcagaagaacttagatcattatataatac agtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaagga agctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagca agcggccactgatcttcagacctggaggaggagatatgagggacaattggagaa gtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggag ctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcctcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaagg atcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactg ctgtgccttggaatgctagttggagtaataaatctctggaacagattggaatca cacgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattatt ggaattagataaatgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaat agtttttgctgtactttctatagtgaatagagttaggcagggatattcaccatt atcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaat agaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcggttaacttttaaaagaaaaggggggattggggggtacag tgcaggggaaagaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaattcaaaattttatcgatactagtattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcg ctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgt tttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtttatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctcc atagaagattctagagccgccaccatgcttctcctggtgacaagccttctgctc tgtgagttaccacacccagcattcctcctgatcccagacattcagcaggtccagctccagcagtctggccctgaactcgaaaaacctggcgctagcgtgaaaatttcc tgtaaagcctccggctactcttttactggctacacaatgaattgggtgaaacag tctcacggcaaatccctcgaatggatcggactcatcacaccctacaatggcgcctcttcctacaaccagaaattccggggcaaggcaacactcactgtggacaaatcatcctctaccgcctacatggatctgctctccctcacatctgaggactccgctgtctacttttgtgcccgaggaggatacgacggacgaggattcgattactggggacag ggaacaactgtgaccgtgtctagtggcggcggagggagtggaggcggaggatcttctggcgggggatccgatattgaactcacacagtctcccgctatcatgtctgct tctcccggcgagaaagtgactatgacttgctctgcttcctcttctgtgtcctac atgcactggtaccagcagaaatctggcacatcccctaaacggtggatctacgat actagcaaactggcatccggcgtgcctgggcgattctctggctctggctctggcaactcttactctctcacaatctcatctgtcgaggctgaggacgatgccacatac tactgtcagcagtggtctaaacacccactcacattcggcgctggcactaaactggaaataaaagcggccgcaggtggcggcggttctggtggcggcggttctggtggcggcggttctctcgaggatggtaatgaagaaatgggtggtattacacagacacca tataaagtctccatctctggaaccacagtaatattgacatgccctcagtatcct ggatctgaaatactatggcaacacaatgataaaaacataggcggtgatgaggat gataaaaacataggcagtgatgaggatcacctgtcactgaaggaattttcagaattggagcaaagtggttattatgtctgctaccccagaggaagcaaaccagaagat gcgaacttttatctctacctgagggcaagagtgtgtgagaactgcatggagatg gatgtgatgtcggtggccacaattgtcatagtggacatctgcatcactgggggcttgctgctgctggtttactactggagcaagaatagaaaggccaaggccaagcctgtgacacgaggagcgggtgctggcggcaggcaaaggggacaaaacaaggagaggccaccacctgttcccaacccagactatgagcccatccggaaaggccagcgggac ctgtattctggcctgaatcagagacgcatctgataagaattcgatccgcggccgcgaaggatctgcgatcgctccggtgcccgtcagtgggcagagcgcacatcgccc acagtccccgagaagttggggggaggggtcggcaattgaacgggtgcctagaga aggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgccttttt cccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctt tttcgcaacgggtttgccgccagaacacagctgaagcttcgaggggctcgcatc tctccttcacgcgcccgccgccctacctgaggccgccatccacgccggttgagtcgcgttctgccgcctcccgcctgtggtgcctcctgaactgcgtccgccgtctag gtaagtttaaagctcaggtcgagaccgggcctttgtccggcgctcccttggagc ctacctagactcagccggctctccacgctttgcctgaccctgcttgctcaactc tacgtctttgtttcgttttctgttctgcgccgttacagatccaagctgtgaccg gcgcctacgctagatgaccgagtacaagcccacggtgcgcctcgccacccgcga cgacgtccccagggccgtacgcaccctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccgccacatcgagcgggtcaccgagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgacggcgccgcggtggcggtctggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagatcggcccgcgcatggccgagttgagcggttcccggctggc cgcgcagcaacagatggaaggcctcctggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcggcgtctcgcccgaccaccagggcaagggtctgggcagcgccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgcaacctccccttctacgagcggctcggctt caccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctggtgcatgac ccgcaagcccggtgcctgagtcgacaatcaacctctggattacaaaatttgtga aagattgactggtattcttaactatgttgctccttttacgctatgtggatacgc tgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctc ctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgt caggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttg gggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccc tattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggc tcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctt tccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctg ctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgcc ggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctc cctttgggccgcctccccgcctggtacctttaagaccaatgacttacaaggcag ctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattc actcccaacgaaaataagatctgctttttgcttgtactgggtctctctggttag accagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggctctagctatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaat tttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaa gtagtgaggaggcttttttggaggcctagacttttgcagagacggcccaaattc gtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattcc acacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagt gagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaa cctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggttt gcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggc caggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccc tgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacagg actataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgt tccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgcctt atccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccact ggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctac agagttcttgaagtggtggcctaactacggctacactagaaggacagtatttgg tatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttg atccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagca gattacacgttaagggattttggtcatgagattatcaaaaaggatcttcaccta gatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagta aacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgat ctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttg ccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgc cattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcag ctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaa agcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagt gttatcactcatggttatggcagcactgcataattctcttactgtcatgccatc cgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaata gtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcg aaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcg tgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagc aaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggtta ttgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaatagg ggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattat tatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtctcgc gcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggt cacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtc agcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagat tgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgc  tgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgccaagctg 17  p510_anti- MLLLVTSLLLCELPHPAFLLIPDIQQVQLQQSGPELEKPGASVKISCKAS  MSLN_SS1_CD GYSFTGYTMNWVKQSFIGKSLEWIGLITPYNGASSYNQKFRGKATLTV  3ϵ amino acid DKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGQGTTVTVSS GGGGSGGGGSSGGGSDIELTQSPAIMSASPGEKVTMTCSASSSVSYMH WYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAED DATYYCQQWSKHPLTFGAGTKLEIKAAAGGGGSGGGGSGGGGSLED GNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDD KNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCE NCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAG AGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI*  18  Anti-MSLN DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQS  Light Chain PKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKITRVEAEDLGVFFCSQSTH  amino acid VPFTFGSGTKLEIK  (MHC1445LC.1)  19  Anti-MSLN gatgttgtgatgacccaaactccactctccctgcctgtcagtcttggagatcaa  Light Chain gcctccatctcttgcagatctagtcagagccttgtacacagtaatggaaacacc  DNA tatttacattggtacctgcagaagccaggccagtctccaaagctcctgatctac  (MHC1445LC.1) aaagtttccaaccgattttctggggtcccagacaggttcagtggcagtggatca gggactgatttcacactcaagatcaccagagtggaggctgaggatctgggagtt tttttctgctctcaaagtacacatgttccattcacgttcggctcggggacaaag ttggaaataaaa 20 Anti-MSLN  QVQLQQSGAELVRPGASVTLSCKASGYTFFDYEMHWVKQTPVHGLE  Heavy Chain WIGAIDPEIDGTAYNQKFKGKAILTADKSSSTAYMELRSLTSEDSAVYY  amino acid CTDYYGSSYWYFDVWGTGTTVTVSS  (MHC1445HC.1)  21  Anti-MSLN caggttcaactgcagcagtctggggctgagctggtgaggcctggggcttcagtg  Heavy Chain acgctgtcctgcaaggcttcgggctacacattttttgactatgaaatgcactgg  DNA gtgaagcagacacctgtgcatggcctggaatggattggagctattgatcctgaa  (MHC1445HC.1) attgatggtactgcctacaatcagaagttcaagggcaaggccatactgactgca gacaaatcctccagcacagcctacatggagctccgcagcctgacatctgaggac tctgccgtctattactgtacagattactacggtagtagctactggtacttcgatgtctggggcacagggaccacggtcaccgtctcctc 22  Anti-MSLN DVMMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWFLQKPGQS  Light Chain PKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQTT  amino acid HVPLTFGAGTKLELK  (MHC1446LC.1)  23  Anti-MSLN gatgttatgatgacccaaactccactctccctgcctgtcagtcttggagatcaa Light Chain gcctccatctcttgcagatctagtcagagccttgtacacagtaatggaaacacc  DNA tatttacattggttcctgcagaagccaggccagtctccaaagctcctgatctac  (MHC1446LC.1) aaagtttccaaccgattttctggggtcccagacaggttcagtggcagtggatca gggacagatttcacactcaagatcagcagagtggaggctgaggatctgggagtt tatttctgctctcaaactacacatgttccgctcacgttcggtgctgggaccaag ctggagctgaaa 24 Anti-MSLN  QVQLQQSGAELVRPGASVTLSCKASGYTFTDYEMHWVKQTPVHGLE  Heavy Chain WIGAIDPEIAGTAYNQKFKGKAILTADKSSSTAYMELRSLTSEDSAVYY  amino acid CSRYGGNYLYYFDYWGQGTTLTVSS  (MHC1446HC.3)  25  Anti-MSLN caggttcaactgcagcagtctggggctgagctggtgaggcctggggcttcagtg  Heavy Chain acgctgtcctgcaaggcttcgggctacacttttactgactatgaaatgcactgg  DNA gtgaagcagacacctgtccatggcctggaatggattggagctattgatcctgaa  (MHC1446HC.3) attgctggtactgcctacaatcagaagttcaagggcaaggccatactgactgca gacaaatcctccagcacagcctacatggagctccgcagcctgacatctgaggactctgccgtctattactgttcaagatacggtggtaactacctttactactttgac tactggggccaaggcaccactctcacagtctcctca 26  Anti-MSLN DVLMTQIPLSLPVSLGDQASISCRSSQNIVYSNGNTYLEWYLQKPGQSP  Light Chain KLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSH  amino acid VPFTFGSGTKLEIK  (MHC1447LC.5)  27  Anti-MSLN gatgttttgatgacccaaattccactctccctgcctgtcagtcttggagatcaa  Light Chain gcctccatctcttgcagatctagtcagaacattgtgtatagtaatggaaacacc  DNA tatttagagtggtacctgcagaaaccaggccagtctccaaagctcctgatctac  (MHC1447LC.5) aaagtttccaaccgattttctggggtcccagacaggttcagtggcagtggatca  gggacagatttcacactcaagatcagcagagtggaggctgaggatctgggagtt  tattactgctttcaaggttcacatgttccattcacgttcggctcggggacaaag ttggaaataaaa 28 Anti-MSLN  QVQLQQSGAELVRPGASVTLSCKASGYTFTDYEMHWVKQTPVHGLE  Heavy Chain WIGAIDPEIGGSAYNQKFKGRAILTADKSSSTAYMELRSLTSEDSAVYY  amino acid CTGYDGYFWFAYWGQGTLVTVSS  (MHC1447HC.5)  29  Anti-MSLN caggttcaactgcagcagtccggggctgagctggtgaggcctggggcttcagtg  Heavy Chain acgctgtcctgcaaggcttcgggctacacatttactgactatgaaatgcactgg  DNA gtgaagcagacacctgtgcatggcctggaatggattggagctattgatcctgaa  (MHC1447HC.5) attggtggttctgcctacaatcagaagttcaagggcagggccatattgactgca gacaaatcctccagcacagcctacatggagctccgcagcctgacatctgaggac tctgccgtctattattgtacgggctatgatggttacttttggtttgcttactggggccaagggactctggtcactgtctcttca 30  Anti-MSLN ENVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSSTSPKLWIY  Light Chain DTSKLASGVPGRFSGSGSGNSYSLTISSMEAEDVATYYCFQGSGYPLTF  amino acid GSGTKLEIK  (MHC1448LC.4)  31  Anti-MSLN gaaaatgttctcacccagtctccagcaatcatgtccgcatctccaggggaaaag  Light Chain gtcaccatgacctgcagtgctagctcaagtgtaagttacatgcactggtaccag  DNA cagaagtcaagcacctcccccaaactctggatttatgacacatccaaactggct  (MHC1448LC.4) tctggagtcccaggtcgcttcagtggcagtgggtctggaaactcttactctctc acgatcagcagcatggaggctgaagatgttgccacttattactgttttcagggg agtgggtacccactcacgttcggctcggggacaaagttggaaataaaa 32  Anti-MSLN QVQLQQSGAELVRPGASVTLSCKASGYTFTDYEMHWVKQTPVHGLE  Heavy Chain WIGGIDPETGGTAYNQKFKGKAILTADKSSSTAYMELRSLTSEDSAVY  amino acid YCTSYYGSRVFWGTGTTVTVSS  (MHC1448HC.3)  33  Anti-MSLN caggttcaactgcagcagtctggggctgagctggtgaggcctggggcttcagtg  Heavy Chain acgctgtcctgcaaggcttcgggctacacatttactgactatgaaatgcactgg  DNA gtgaaacagacacctgtgcatggcctggaatggattggaggtattgatcctgaa  (MHC1448HC.3) actggtggtactgcctacaatcagaagttcaagggtaaggccatactgactgcagacaaatcctccagcacagcctacatggagctccgcagcctgacatctgaggac tctgccgtctattactgtacaagttactatggtagtagagtcttctggggcacagggaccacggtcaccgtctcctca 34  Anti-MSLN QIVLSQSPAILSAFPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIY  Light Chain ATSNLASGVPARFSGSGSGTSYSLTISSVEAEDAATYYCQQWSSNPPTL  amino acid TFGAGTKLELK  (MHC1449LC.3)  35  Anti-MSLN caaattgttctctcccagtctccagcaatcctgtctgcatttccaggggagaag Light Chain gtcactatgacttgcagggccagctcaagtgtaagttacatgcactggtaccag  DNA cagaagccaggatcctcccccaaaccctggatttatgccacatccaacctggct  (MHC1449LC.3) tctggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctc acaatcagcagtgtggaggctgaagatgctgccacttattactgccagcagtgg agtagtaacccacccacgctcacgttcggtgctgggaccaagctggagctgaaa 36  Anti-MSLN QVQLQQSGAELARPGASVKLSCKASGYTFTSYGISWVKQRTGQGLEW  Heavy Chain IGEIYPRSGNTYYNESFKGKVTLTADKSSGTAYMELRSLTSEDSAVYFC  amino acid ARWGSYGSPPFYYGMDYWGQGTSVTVSS  (MHC1449HC.3)  37  Anti-MSLN caggttcagctgcagcagtctggagctgagctggcgaggcctggggcttcagtg  Heavy Chain aagctgtcctgcaaggcttctggctacaccttcacaagctatggtataagctgg  DNA gtgaagcagaggactggacagggccttgagtggattggagagatttatcctaga  (MHC1449HC.3) agtggtaatacttactacaatgagagcttcaagggcaaggtcacactgaccgca gacaaatcttccggcacagcgtacatggagctccgcagcctgacatctgaggac tctgcggtctatttctgtgcaagatggggctcctacggtagtccccccttttactatggtatggactactggggtcaaggaacctcagtcaccgtctcctca 38  Anti-MSLN DVLMTQTPLSLPVSLGNQASISCRSSQSIVHSSGSTYLEWYLQKPGQSP  Light Chain KLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSH  amino acid VPYTFGGGTKLEIK  (MHC1450LC.3)  39  Anti-MSLN gatgttttgatgacccaaactccactctccctgcctgtcagtcttggaaatcaag  Light Chain cctccatctcttgcagatctagtcagagcattgtacatagtagtggaagcaccta  DNA tttagaatggtacctgcagaaaccaggccagtctccaaagctcctgatctacaaa  (MHC1450LC.3) gtttccaaccgattttctggggtcccagacaggttcagtggcagtggatcaggga cagatttcacactcaagatcagcagagtggaggctgaggatctgggagtttatta ctgctttcaaggctcacatgttccatacacgttcggaggggggaccaagctggaa ataaaa 40 Anti-MSLN  QVQLQQSGAELARPGTSVKVSCKASGYTFTSYGISWVKQRIGQGLEWI Heavy Chain  GEIHPRSGNSYYNEKIRGKATLTADKSSSTAYMELRSLISEDSAVYFCA amino acid  RLITTVVANYYAMDYWGQGTSVTVSS  (MHC1450HC.5)  41  Anti-MSLN caggttcagctgcagcagtctggagctgagctggcgaggcctgggacttcagtga  Heavy Chain aggtgtcctgcaaggcttctggctataccttcacaagttatggtataagctgggt  DNA gaagcagagaattggacagggccttgagtggattggagagattcatcctagaagt  (MHC1450HC.5) ggtaatagttactataatgagaagatcaggggcaaggccacactgactgcagaca aatcctccagcacagcgtacatggagctccgcagcctgatatctgaggactctgc ggtctatttctgtgcaaggctgattactacggtagttgctaattactatgctatggactactggggtcaaggaacctcagtcaccgtctcctca 42  Anti-MSLN DIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPG  Light Chain QSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQ  amino acid SYNLVTFGAGTKLELK  (MHC1451LC.1)  43  Anti-MSLN gacattgtgatgtcacagtctccatcctccctggctgtgtcagcaggagagaagg  Light Chain tcactatgagctgcaaatccagtcagagtctgctcaacagtagaacccgaaagaa  DNA ctacttggcttggtaccagcagaaaccagggcagtctcctaaactgctgatctac  (MHC1451LC.1) tgggcatccactagggaatctggggtccctgatcgcttcacaggcagtggatctg ggacagatttcactctcaccatcagcagtgtgcaggctgaagacctggcagttta ttactgcaaacaatcttataatctggtcacgttcggtgctgggaccaagctggag ctgaaa 44 Anti-MSLN  QVQLQQSGAELVRPGASVTLSCKASGYTFFDYEMHWVKQTPVHGLE  Heavy Chain WIGAIDPEIDGTAYNQKFKGKAILTADKSSSTAYMELRSLTSEDSAVYY  amino acid CTDYYGSSYWYFDVWGTGTTVTVSS  (MHC1451HC.2)  45  Anti-MSLN caggttcaactgcagcagtctggggctgagctggtgaggcctggggcttcagtga  Heavy Chain cgctgtcctgcaaggcttcgggctacacattttttgactatgaaatgcactgggt  DNA gaagcagacacctgtgcatggcctggaatggattggagctattgatcctgaaatt  (MHC1451HC.2) gatggtactgcctacaatcagaagttcaagggcaaggccatactgactgcagaca aatcctccagcacagcctacatggagctccgcagcctgacatctgaggactctgc cgtctattactgtacagattactacggtagtagctactggtacttcgatgtctggggcacagggaccacggtcaccgtctcctc 46  Anti-MSLN QIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSPKPWIYR  Light Chain TSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPLTFG  amino acid  AGTKLELK (MHC1452LC.1)  47  Anti-MSLN caaattgttctcacccagtctccagcaatcatgtctgcatctccaggggagaagg  Light Chain tcaccatatcctgcagtgccagctcaagtgtaagttacatgtactggtaccagca  DNA gaagccaggatcctcccccaaaccctggatttatcgcacatccaacctggcttct  (MHC1452LC. 1) ggagtccctgctcgcttcagtggcagtgggtctgggacctcttactctctcacaa tcagcagcatggaggctgaagatgctgccacttattactgccagcagtatcatag ttacccactcacgttcggtgctgggaccaagctggagctgaaa 48  Anti-MSLN QIVLTQSPAIMSASPGERVTMTCSASSSVSSSYLYWYQQKSGSSPKLWI  Light Chain YSISNLASGVPARFSGSGSGTSYSLTINSMEAEDAATYYCQQWSSNPQL  amino acid TFGAGTKLELK  (MHC1452LC.6)  49  Anti-MSLN caaattgttctcacccagtctccagcaatcatgtctgcatctcctggggaacggg  Light Chain tcaccatgacctgcagtgccagctcaagtgtaagttccagctacttgtactggta  DNA ccagcagaagtcaggatcctccccaaaactctggatttatagcatatccaacctg  (MHC1452LC.6) gcttctggagtcccagctcgcttcagtggcagtgggtctgggacctcttactctc tcacaatcaacagcatggaggctgaagatgctgccacttattactgccagcagtggagtagtaacccacagctcacgttcggtgctgggaccaagctggagctgaaa 56  Anti-MSLN QVQLKQSGAELVKPGASVKISCKASGYTFTDYYINWVKQRPGQGLEW  Heavy Chain IGKIGPGSGSTYYNEKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFC  amino acid ARTGYYVGYYAMDYWGQGTSVTVSS  (MHC1452HC.2)  50  Anti-MSLN caggtccagctgaagcagtctggagctgagctggtgaagcctggggcttcagtga  Heavy Chain agatatcctgcaaggcttctggctacaccttcactgactactatataaactgggt  DNA gaagcagaggcctggacagggccttgagtggattggaaagattggtcctggaagt  (MHC1452HC.2) ggtagtacttactacaatgagaagttcaagggcaaggccacactgactgcagaca aatcctccagcacagcctacatgcagctcagcagcctgacatctgaggactctgcagtctatttctgtgcaagaactggttactacgttggttactatgctatggactac tggggtcaaggaacctcagtcaccgtctcctca 51  Anti-MSLN QVQLQQSGAELARPGASVKLSCKASGYTFTIYGISWVKQRTGQGLEWI  Heavy Chain GEIYPRSDNTYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFC  amino acid ARWYSFYAMDYWGQGTSVTVSS  (MHC1452HC.4)  52  Anti-MSLN caggttcagctgcagcagtctggagctgagctggcgaggcctggggcttcagtg  Heavy Chain aagctgtcctgcaaggcttctggctacaccttcacaatctatggtataagctgg  DNA gtgaaacagagaactggacagggccttgagtggattggagagatttatcctaga  (MHC1452HC.4) agtgataatacttactacaatgagaagttcaagggcaaggccacactgactgcagacaaatcctccagcacagcgtacatggagctccgcagcctgacatctgaggactctgcggtctatttctgtgcaagatggtactcgttctatgctatggactactggggtcaaggaacctcagtcaccgtctcctca  58  Single domain EVQLVESGGGLVQPGGSLRLSCAASGGDWSANFMYWYRQAPGKQRE  anti-MSLN LVARISGRGVVDYVESVKGRFTISRDNSKNTLYLQMNSLRAEDTAVY  binder 1 (SD1) YCAVASYWGQGTLVTVSS  59  Single domain EVQLVESGGGLVQPGGSLRLSCAASGSTSSINTMYWYRQAPGKERELV  anti-MSLN AFISSGGSTNVRDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCN  binder 4 (SD4) TYIPYGGTLHDFWGQGTLVTVSS  55  Single domain QVQLVESGGGVVQAGGSLRLSCAASGSTFSIRAMRWYRQAPGTERDL  anti-MSLN VAVIYGSSTYYADAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC  binder 6 (SD6) NADTIGTARDYWGQGTLVTVSS 

What is claimed is:
 1. A recombinant nucleic acid molecule encoding a Tcell receptor (TCR) fusion protein (TFP) comprising: (a) a TCR subunitcomprising (i) an extracellular domain of a TCR subunit or a portionthereof, (ii) a transmembrane domain, and (iii) a TCR intracellulardomain comprising a stimulatory domain from an intracellular signalingdomain of CD3 epsilon or CD3 gamma; and b) a human or humanized antibodydomain comprising an antigen binding domain that is an anti-mesothelinsingle domain antibody comprising SEQ ID NO:58 or SEQ ID NO:59; whereinthe TCR subunit and the antibody domain are operatively linked; andwherein the TFP incorporates into a functional TCR when expressed in a Tcell.
 2. The recombinant nucleic acid molecule of claim 1, wherein theTCR subunit comprises at least a portion of CD3 epsilon.
 3. Therecombinant nucleic acid molecule of claim 2, wherein the TCR subunitcomprises an amino acid sequence that is at least 85% identical to SEQID NO:
 4. 4. The recombinant nucleic acid molecule of claim 2, whereinthe TCR subunit comprises an amino acid sequence that is at least 89%identical to SEQ ID NO:
 4. 5. The recombinant nucleic acid molecule ofclaim 1, wherein the TCR subunit comprises a CD3 epsilon extracellulardomain or a portion thereof.
 6. The recombinant nucleic acid molecule ofclaim 5, wherein the CD3 epsilon extracellular domain or portion thereofhas an amino acid sequence identical to amino acids 23-126 of SEQ ID NO:4.
 7. The recombinant nucleic acid molecule of claim 1, wherein the TCRsubunit comprises a CD3 epsilon transmembrane domain.
 8. The recombinantnucleic acid molecule of claim 7, wherein the CD3 epsilon transmembranedomain has an amino acid sequence identical to amino acids 127-152 ofSEQ ID NO:
 4. 9. The recombinant nucleic acid molecule of claim 1,wherein the TCR subunit comprises a CD3 epsilon stimulatory domain. 10.The recombinant nucleic acid molecule of claim 9, wherein the CD3epsilon stimulatory domain is an immunoreceptor tyrosine-basedactivation motif (ITAM) having an amino acid sequence identical to aminoacids 178-205 of SEQ ID NO:
 4. 11. The recombinant nucleic acid moleculeof claim 1, wherein the TCR subunit comprises a CD3 epsilonintracellular domain.
 12. The recombinant nucleic acid molecule of claim11, wherein the CD3 epsilon intracellular domain has an amino acidsequence identical to positions 153-207 of SEQ ID NO:
 4. 13. Therecombinant nucleic acid molecule of claim 1, wherein theanti-mesothelin single domain antibody is a V_(HH) domain.
 14. Therecombinant nucleic acid molecule of claim 1, wherein theanti-mesothelin single domain antibody comprises SEQ ID NO:58.
 15. Therecombinant nucleic acid molecule of claim 1, wherein the human orhumanized antibody domain is linked to the N-terminus of the TCR subunitby a linker.
 16. The recombinant nucleic acid molecule of claim 15,wherein the linker comprises the sequence of (G₄S)_(n), wherein G isglycine, S is serine, and n is an integer from 1 to
 4. 17. Therecombinant nucleic acid molecule of claim 15, wherein the linkercomprises SEQ ID NO:
 2. 18. The recombinant nucleic acid molecule ofclaim 1, wherein the TFP further comprises a leader sequence.
 19. Therecombinant nucleic acid molecule of claim 18, wherein the leadersequence comprises an amino acid sequence identical to amino acids 1-22of SEQ ID NO:
 17. 20. A T cell comprising a recombinant nucleic acidmolecule encoding a T cell receptor (TCR) fusion protein (TFP)comprising (a) a TCR subunit comprising (i) an extracellular domain of aTCR subunit or a portion thereof, (ii) a transmembrane domain, and (iii)a TCR intracellular domain comprising a stimulatory domain from anintracellular signaling domain of CD3 epsilon or CD3 gamma; and b) ahuman or humanized antibody domain comprising an antigen binding domainthat is an anti-mesothelin single domain antibody comprising SEQ IDNO:58 or SEQ ID NO:59; wherein the TCR subunit and the antibody domainare operatively linked; and wherein the TFP incorporates into afunctional TCR when expressed in the T cell.
 21. The T cell of claim 20,wherein the TCR subunit comprises at least a portion of CD3 epsilon. 22.The T cell of claim 20, wherein the T cell is selected from a human CD8+T cell, a human CD4+ T cell, a human gamma-delta T cell, a human NKTcell, and combinations thereof.
 23. The T cell of claim 20, whereinproduction of a cytokine by the T cell is lower than production of thecytokine by a T cell comprising a nucleic acid encoding a CAR comprisingthe antigen binding domain operatively linked to at least a portion of aCD28 extracellular domain, a CD28 transmembrane domain, at least aportion of a CD28 intracellular domain, and a CD3 zeta intracellulardomain.
 24. The T cell of claim 23, wherein the cytokine is IL-2, IFNγ,or a combination thereof.
 25. The T cell claim 20, wherein production ofIL-2 by the T cell is increased in the presence of a human cellexpressing mesothelin compared to a T cell not containing the TFP. 26.The T cell of claim 20, wherein the T cell exhibits increasedcytotoxicity to a human cell expressing mesothelin compared to a T cellnot containing the TFP.
 27. A pharmaceutical composition comprising a Tcell from a human subject, wherein the T cell comprises a recombinantnucleic acid molecule encoding a T cell receptor (TCR) fusion protein(TFP) comprising: (a) a TCR subunit comprising (i) a CD3 epsilonextracellular domain having a sequence identical to amino acids 23-126of SEQ ID NO: 4, (ii) a CD3 epsilon transmembrane domain having asequence identical to amino acids 127-152 SEQ ID NO: 4, and (iii) a CD3epsilon intracellular domain having a sequence identical to amino acids153-207 of SEQ ID NO: 4; and b) an anti-mesothelin single domainantibody comprising SEQ ID NO:58; and wherein the anti-mesothelin singledomain antibody is linked to the N-terminus of the TCR subunit by alinker comprising SEQ ID NO: 2.